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  • 1.
    Boström, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Markland, Katrin
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Sandén, Anna Maria
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli2005Ingår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, nr 1, s. 82-90Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

  • 2.
    Dekki Shalaly, Nancy
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ria, Massimiliano
    Johansson, Ulrika
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Avall, Karin
    Berggren, Per-Olof
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Silk matrices promote formation of insulin-secreting islet-like clusters2016Ingår i: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 90, s. 50-61Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation.

  • 3. Fuentes, L.
    et al.
    Gomez-Cid, L.
    Fernandez-Santos, M. E.
    Suarez-Sancho, S.
    Plasencia, V.
    Climent, A.
    Sanz-Ruiz, R.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Protein-based materials.
    Atienza, F.
    Fernandez-Aviles, F.
    Biocompatibility of mesenchymal stem cells with a spider silk matrix and its potential use as scaffold for cardiac tissue regeneration2016Ingår i: Cardiovascular Research, ISSN 0008-6363, E-ISSN 1755-3245, Vol. 111, s. S93-S93Artikel i tidskrift (Övrigt vetenskapligt)
  • 4.
    Gomez-Cid, L.
    et al.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Fuentes, L.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Fernandez-Santos, M. E.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Suarez-Sancho, S.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Plasencia, V.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Climent, A. M.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Sanz-Ruiz, R.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Atienza, F.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Aviles, F. F.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Effect of spider silk matrix on cardiac tissue regeneration of mesenchymal stem cells2018Ingår i: European Journal of Clinical Investigation, ISSN 0014-2972, E-ISSN 1365-2362, Vol. 48, s. 150-150Artikel i tidskrift (Övrigt vetenskapligt)
  • 5.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Ehn, Maria
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Gunnel, Lundin
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hedhammar, My
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner2002Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, nr 1-2, s. 157-166Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

  • 6.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Hedhammar, My
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 93-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 7.
    Guo, Weijin
    et al.
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Mikro- och nanosystemteknik.
    Gustafsson, Linnea
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Mikro- och nanosystemteknik.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    van der Wijngaart, Wouter
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Mikro- och nanosystemteknik.
    Formation of a thin-walled Spider Silk Tube on a Micromachined Scaffold2018Ingår i: Proceeding of 2018 IEEE 31st International Conference on Micro Electro Mechanical Systems (MEMS), Institute of Electrical and Electronics Engineers (IEEE), 2018, Vol. 2018, s. 83-85Konferensbidrag (Refereegranskat)
    Abstract [en]

    This paper reports on the first formation of a thin bio-functionalized spider silk tube, supported by an internal micromachined scaffold, in which both the inside and outside of the tube wall are freely accessible. The silk tube could potentially be used as an artificial blood vessel in an in vitro tissue scaffold, where endothelial cells and tissue cells can grow on both sides of the silk tube.

  • 8.
    Güler, Rezan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Thatikonda, Naresh
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Ghani, Hawraa Ali
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider SilkManuskript (preprint) (Övrigt vetenskapligt)
  • 9.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Strategies for facilitated protein recovery after recombinant production in Escherichia coli2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.

    A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger.

    Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography

    Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer.

    The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin.

  • 10.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Alm, Tove
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag2006Ingår i: Biotechnology Journal, ISSN 1860-6768, Vol. 1, s. 187-196Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

  • 11.
    Hedhammar, My
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Gräslund, Torbjörn
    KTH, Tidigare Institutioner, Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner, Bioteknologi.
    Negatively charged purification tags for selective anion-exchange recovery2004Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, nr 11, s. 779-786Artikel i tidskrift (Refereegranskat)
    Abstract [en]

     A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

  • 12.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Z(basic) - A novel purification tag for efficient protein recovery2007Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1161, nr 1-2, s. 22-28Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A positively charged protein domain, Z(basic) can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z(basic) was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z(basic) as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z(basic), tag in small and pilot-plant-scale downstream processing.

  • 13.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Jung, H. R.
    University of Southern Denmark, Department for Biochemistry and Molecular Biology.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Enzymatic cleavage of fusion proteins using immobilised protease 3C2006Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 47, nr 2, s. 422-426Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A strategy for efficient cleavage of fusion proteins using an immobilised protease has been developed. Protease 3C from coxsackie virus was recombinantly produced in Escherichia coli and covalently immobilised onto a solid support. Thereafter, Z(basic) tagged fusion proteins, with a specific cleavage sequence between the domains, were flown through the proteolytic column and circulated until complete cleavage. Subsequently, the processed protein solution was applied on a cation exchanger. Thereby, removal of the released, positively charged fusion tag, Z(basic), was done by adsorption to the matrix while the target proteins were recovered in the flow through. Interestingly, the columns were shown to be reusable without any measurable decrease in activity. Moreover, after storage in 4 degrees C for two months the activity was almost unaffected.

  • 14.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Zbasic: a purification tag for selective ion-exchange recovery2014Ingår i: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1129, s. 197-204Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived domain and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchange resin and thereby allow solid-phase refolding of the target protein.Herein, protocols for purification of soluble Zbasic-tagged fusion proteins, as well as for integrated purification and solid-phase refolding of insoluble fusion proteins, are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.

  • 15.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lönneborg, Rosa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nord, Olof
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sjölin, Olle
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Uhlén, Matthias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein2005Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, nr 2, s. 133-146Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

  • 16.
    Horak, Josef
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Dev, Apurba
    Uppsala Univ, Ångström Lab, Solid State Elect, Uppsala Box 534, SE-75121 Uppsala, Sweden..
    Nilebäck, Linnea
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Behnam, Kiarash
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Linnros, Jan
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Fotonik.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Recombinant Spider Silk as Mediator for One-Step, Chemical-Free Surface Biofunctionalization2018Ingår i: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 28, nr 21, artikel-id 1800206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A unique strategy for effective, versatile, and facile surface biofunctionalization employing a recombinant spider silk protein genetically functionalized with the antibody-binding Z domain (Z-4RepCT) is reported. It is demonstrated that Z-silk can be applied to a variety of materials and platform designs as a truly one-step and chemical-free surface modification that site specifically captures antibodies while simultaneously reducing nonspecific adsorption. As a model surface, SiO2 is used to optimize and characterize Z-silk performance compared to the Z domain immobilized by a standard silanization method. First, Z-silk adsorption is investigated and verified its biofunctionality in a long-term stability experiment. To assess the binding capacity and protein-protein interaction stability of Z-silk, the coating is used to capture human antibodies in various assay formats. An eightfold higher binding capacity and 40-fold lower detection limit are obtained in the immunofluorescence assay, and the complex stability of captured antibodies is shown to be improved by a factor of 20. Applicability of Z-silk to functionalize microfluidic devices is demonstrated by antibody detection in an electrokinetic microcapillary biosensor. To test Z-silk for biomarker applications, real-time detection and quantification of human immunoglobulin G are performed in a plasma sample and C1q capture from human serum using an anti-C1q antibody.

  • 17. Jansson, R.
    et al.
    Lau, C. H.
    Ishida, T.
    Ramström, M.
    Sandgren, M.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Swedish University of Agricultural Sciences, Sweden.
    Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris2016Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 11, nr 5, s. 687-699Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.

  • 18. Jansson, Ronnie
    et al.
    Courtin, Christophe M.
    Sandgren, Mats
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Swedish University of Agricultural Sciences, Sweden.
    Rational Design of Spider Silk Materials Genetically Fused with an Enzyme2015Ingår i: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 25, nr 33, s. 5343-5352Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Enzyme immobilization is an attractive route for achieving catalytically functional surfaces suitable for both continuous and repeated use. Herein, genetic engineering is used to combine the catalytic ability of a xylanase with the self-assembly properties of recombinant spider silk, realizing silk materials with enzymatic activity. Under near-physiological conditions, soluble xylanase-silk fusion proteins assembled into fibers displaying catalytic activity. Also, a xylanase-silk protein variant with the silk part miniaturized to contain only the C-terminal domain of the silk protein formed fibers with catalytic activity. The repertoire of xylanase-silk formats is further extended to include 2D surface coatings and 3D foams, also being catalytically active, showing the versatile range of possible silk materials. The stability of the xylanase-silk materials is explored, demonstrating the possibility of storage, reuse, and cleaning with ethanol. Interestingly, fibers can also be stored dried with substantial residual activity after rehydration. Moreover, a continuous enzymatic reaction using xylanase-silk is demonstrated, making enzymatic batch reactions not the sole possible implementation. The proof-of-concept for recombinantly produced enzyme-silk, herein shown with a xylanase, implies that also other enzymes can be used in similar setups. It is envisioned that the concept of enzyme-silk can find its applicability in, for example, multienzyme reaction systems or biosensors.

  • 19. Johansson, Ulrika
    et al.
    Ria, Massimiliano
    Avall, Karin
    Shalaly, Nancy Dekki
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Zaitsev, Sergei V.
    Berggren, Per-Olof
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 6, artikel-id e0130169Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added.

  • 20.
    Johansson, Ulrika
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. Linnæus Center of Biomaterials Chemistry, Linnæus University, Kalmar, Sweden Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Widhe, Mona
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Shalaly, Nancy Dekki
    Arregui, Irene Linares
    Nilebäck, Linnea
    Tasiopoulos, Christos Panagiotis
    Åstrand, Carolina
    Berggren, Per-Olof
    Gasser, Christian
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers.2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, nr 1, artikel-id 6291Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.

  • 21. Landreh, M.
    et al.
    Askarieh, G.
    Nordling, K.
    Hedhammar, My
    Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, SE-751 23 Uppsala, Sweden.
    Rising, A.
    Casals, C.
    Astorga-Wells, J.
    Alvelius, G.
    Knight, S. D.
    Johansson, J.
    Jornvall, H.
    Bergman, T.
    A pH-dependent dimer lock in spider silk protein2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 404, nr 2, s. 328-336Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below--but not above--6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.

  • 22.
    Mittal, Nitesh
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för teknikvetenskap (SCI), Centra, Linné Flow Center, FLOW. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Jansson, Ronnie
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Widhe, Mona
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Benselfelt, Tobias
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center. Innventia AB, Sweden.
    Håkansson, Karl M. O.
    KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för teknikvetenskap (SCI), Centra, Linné Flow Center, FLOW.
    Lundell, Fredrik
    KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för teknikvetenskap (SCI), Centra, Linné Flow Center, FLOW. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Söderberg, Daniel
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för teknikvetenskap (SCI), Centra, Linné Flow Center, FLOW. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Ultrastrong and Bioactive Nanostructured Bio-Based Composites2017Ingår i: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 11, nr 5, s. 5148-5159Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nature’s design of functional materials relies on smart combinations of simple components to achieve desired properties. Silk and cellulose are two clever examples from nature–spider silk being tough due to high extensibility, whereas cellulose possesses unparalleled strength and stiffness among natural materials. Unfortunately, silk proteins cannot be obtained in large quantities from spiders, and recombinant production processes are so far rather expensive. We have therefore combined small amounts of functionalized recombinant spider silk proteins with the most abundant structural component on Earth (cellulose nanofibrils (CNFs)) to fabricate isotropic as well as anisotropic hierarchical structures. Our approach for the fabrication of bio-based anisotropic fibers results in previously unreached but highly desirable mechanical performance with a stiffness of ∼55 GPa, strength at break of ∼1015 MPa, and toughness of ∼55 MJ m–3. We also show that addition of small amounts of silk fusion proteins to CNF results in materials with advanced biofunctionalities, which cannot be anticipated for the wood-based CNF alone. These findings suggest that bio-based materials provide abundant opportunities to design composites with high strength and functionalities and bring down our dependence on fossil-based resources.

  • 23.
    Mittal, Nitesh
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Lundell, Fredrik
    KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Wågberg, Lars
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Hedhammar, My
    Söderberg, Daniel
    KTH, Skolan för teknikvetenskap (SCI), Mekanik. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Wood Science Center.
    Flow-assisted organization of nanostructured bio-based materials2018Ingår i: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 255Artikel i tidskrift (Övrigt vetenskapligt)
  • 24.
    Nilebäck, Linnea
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Arola, Suvi
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Kvick, Mathias
    AlbaNova Univ Ctr, Spiber Technol AB, S-10691 Stockholm, Sweden..
    Paananen, Arja
    VTT Tech Res Ctr Finland Ltd, Tietotie 2, FI-02150 Espoo, Finland..
    Linder, Markus B.
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism2018Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, nr 39, s. 11795-11805Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mechanism of silk assembly, and thus the cues for the extraordinary properties of silk, can be explored by studying the simplest protein parts needed for the formation of silk-like materials. The recombinant spider silk protein 4RepCT, consisting of four repeats of polyalanine and glycine-rich segments (4Rep) and a globular C-terminal domain (CT), has previously been shown to assemble into silk-like fibers at the liquid-air interface. Herein, we study the interfacial behavior of the two parts of 4RepCT, revealing new details on how each protein part is crucial for the silk assembly. Interfacial rheology and quartz crystal microbalance with dissipation show that 4Rep interacts readily at the interfaces. However, organized nanofibrillar structures are formed only when 4Rep is fused to CT. A strong interplay between the parts to direct the assembly is demonstrated. The presence of either a liquid-air or a liquid-solid interface had a surprisingly similar influence on the assembly.

  • 25.
    Nilebäck, Linnea
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Chouhan, Dimple
    Jansson, Ronnie
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Widhe, Mona
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Mandal, Biman B.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials2017Ingår i: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 9, nr 37, s. 31634-31644Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrixes. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains. The coating process is based on a silk assembly to achieve stable interactions between the silk types under mild buffer conditions. The assembly process was studied in real time by quartz crystal microbalance with dissipation. Coatings, electrospun mats, and microporous scaffolds were constructed from Antheraea assama and Bombyx mori SFs. The morphology of the fibroin materials before and after coating with recombinant silk proteins was analyzed by scanning electron microscopy and atomic force microscopy. SF materials coated with various bioactive 4RepCT fusion proteins resulted in directed antibody capture, enzymatic activity, and improved cell attachment and spreading, respectively, compared to pristine SF materials. The herein-described procedure allows a fast and easy route for the construction of bioactive materials.

  • 26.
    Nilebäck, Linnea
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hedin, Jesper
    Widhe, Mona
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Floderus, Lotta S.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Krona, Annika
    Bysell, Helena
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study2017Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 18, nr 3, s. 846-854Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Functionalization of biomaterials with biologically active peptides can improve their performance after implantation. By genetic fusion to self-assembling proteins, the functional peptides can easily be presented on different physical formats. Herein, a chemical-free coating method based on self-assembly of the recombinant spider silk protein 4RepCT is described and used to prepare functional coatings on various biomaterial surfaces. The silk assembly was studied in real-time, revealing the occurrence of continuous assembly of silk proteins onto surfaces and the formation of nanofibrillar structures. The adsorbed amounts and viscoelastic properties were evaluated, and the coatings were shown to be stable against wash with hydrogen chloride, sodium hydroxide, and ethanol. Titanium, stainless steel, and hydroxyapatite were coated with silk fused to an antimicrobial peptide or a motif from fibronectin. Human primary cells cultured on the functional silk coatings show good cell viability and proliferation, implying the potential to improve implant performance and acceptance by the body.

  • 27.
    Nilebäck, Linnea
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Widhe, Mona
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Seijsing, Johan
    Bysell, Helena
    Sharma, Prashant K.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Bioactive silk coatings reduce adhesion of Staphylococcus aureus while supporting growth of osteoblast-like cellsManuskript (preprint) (Övrigt vetenskapligt)
  • 28.
    Petrou, Georgia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Hogqvist, Mark
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Erlandsson, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Wågberg, Lars
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Genetically Engineered Mucoadhesive Spider Silk2018Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, nr 8, s. 3268-3279Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mucoadhesion is defined as the adhesion of a material to the mucus gel covering the mucous membranes. The mechanisms controlling mucoadhesion include nonspecific electrostatic interactions and specific interactions between the materials and the mucins, the heavily glycosylated proteins that form the mucus gel. Mucoadhesive materials can be used to develop mucosal wound dressings and noninvasive transmucosal drug delivery systems. Spider silk, which is strong, biocompatible, biodegradable, nontoxic, and lightweight would serve as an excellent base for the development of such materials. Here, we investigated two variants of the partial spider silk protein 4RepCT genetically engineered in order to functionalize them with mucoadhesive properties. The pLys-4RepCT variant was functionalized with six cationically charged lysines, aiming to provide nonspecific adhesion from electrostatic interactions with the anionically charged mucins, while the hGal3-4RepCT variant was genetically fused with the Human Galectin-3 Carbohydrate Recognition Domain which specifically binds the mucin glycans Gal beta 1-3GlcNAc and Gal beta 1-4GlcNAc. First, we demonstrated that coatings, fibers, meshes, and foams can be readily made from both silk variants. Measured by the adsorption of both bovine submaxillary mucin and pig gastric mucin, the newly produced silk materials showed enhanced mucin binding properties compared with materials of wild-type (4RepCT) silk. Moreover, we showed that pLys-4RepCT silk coatings bind mucins through electrostatic interactions, while hGal3-4RepCT silk coatings bind mucins through specific glycan-protein interactions. We envision that the two new mucoadhesive silk variants pLys-4RepCT and hGal3-4RepCT, alone or combined with other biofunctional silk proteins, constitute useful new building blocks for a range of silk protein-based materials for mucosal treatments.

  • 29.
    Petrou, Georgia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. KTH Royal Inst Technol, Div Prot Technol, Stockholm, Sweden..
    Högqvist, Mark
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap. KTH Royal Inst Technol, Div Glycosci, Stockholm, Sweden..
    Engineering mucoadhesive silk2018Ingår i: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 255Artikel i tidskrift (Övrigt vetenskapligt)
  • 30.
    Ramachandraiah, Harisha
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Kugiejko, Karol
    KTH, Skolan för bioteknologi (BIO).
    Pettersson, Torbjörn
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi.
    Heuchel, Rainer
    Karolinska Institutet.
    Löhr, Matthias
    karolinska Institute.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Russom, Aman
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Microfluidic based circulating tumor cell isolation and release from whole blood of pancreatic cancer patients using bio-functionalized recombinant spider silkManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    A bio-functionalized microsystem was developed for the capture and release of cancer cells from whole blood. Effective isolation and purification of circulating tumor cells from whole blood provides important capability for clinical application and biological research. Here, we demonstrate a single step surface modification procedure for a microfluidic device based on self-assembly of recombinant spider silk harbouring an affinity domain for antibody binding. The surfaces of microfluidic devices were conjugated/equipped with anti-EpCAM antibody for selective isolation of pancreatic cancer cells from spiked whole blood and finally circulating tumor cells from pancreatic cancer patients. Moreover, a protease-cleavage site in the recombinant spider silk proteins provides the unique option to release the captured cancer cells on command from the device without compromising the cell’s viability. Our approach offers a simple, easy and robust surface modification process with a 85% cancer cell capture efficiency. Subsequent addition of a site-specific protease results in the release of 95% of captured cells from the bio functionalized microfluidic systems. 

  • 31. Rising, A.
    et al.
    Widhe, M.
    Johansson, J.
    Hedhammar, My
    Department of Anatomy Physiology and Biochemistry, Temp Medical Centre, Swedish University of Agricultural Sciences, Uppsala .
    Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications2011Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 68, nr 2, s. 169-84Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Spider dragline silk is an outstanding material made up of unique proteins-spidroins. Analysis of the amino acid sequences of full-length spidroins reveals a tripartite composition: an N-terminal non-repetitive domain, a highly repetitive central part composed of approximately 100 polyalanine/glycine rich co-segments and a C-terminal non-repetitive domain. Recent molecular data on the terminal domains suggest that these have different functions. The composite nature of spidroins allows for recombinant production of individual and combined regions. Miniaturized spidroins designed by linking the terminal domains with a limited number of repetitive segments recapitulate the properties of native spidroins to a surprisingly large extent, provided that they are produced and isolated in a manner that retains water solubility until fibre formation is triggered. Biocompatibility studies in cell culture or in vivo of native and recombinant spider silk indicate that they are surprisingly well tolerated, suggesting that recombinant spider silk has potential for biomedical applications.

  • 32. Stark, M.
    et al.
    Grip, S.
    Rising, A.
    Hedhammar, My
    Department of Anatomy, Physiology, and Biochemistry, The Biomedical Centre, Swedish University of Agricultural Sciences, SE-751 23 Uppsala, Sweden.
    Engstrom, W.
    Hjalm, G.
    Johansson, J.
    Macroscopic fibers self-assembled from recombinant miniature spider silk proteins2007Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, nr 5, s. 1695-1701Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Strength, elasticity, and biocompatibility make spider silk an attractive resource for the production of artificial biomaterials. Spider silk proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline silks, and although self-assembled from recombinant proteins they are as strong as fibers spun from redissolved silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions.

  • 33.
    Tasiopoulos, Christos Panagiotis
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Widhe, Mona
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Recombinant Spider Silk Functionalized with a Motif from Fibronectin Mediates Cell Adhesion and Growth on Polymeric Substrates by Entrapping Cells During Self-Assembly2018Ingår i: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, nr 17, s. 14531-14539Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In vitro endothelialization of synthetic grafts or engineered vascular constructs is considered a promising alternative to overcome shortcomings in the availability of autologous vessels and in graft complications with synthetics. A number of cell-seeding techniques have been implemented to render vascular grafts accessible for cells to attach, proliferate, and spread over the surface area. Nonetheless, seeding efficiency and the time needed for cells to adhere varies dramatically. Herein, we investigated a novel cell-seeding approach (denoted co-seeding) that enables cells to bind to a motif from fibronectin included in a recombinant spider silk protein. Entrapment of cells occurs at the same time as the silk assembles into a nanofibrillar coating on various substrates. Cell adhesion analysis showed that the technique can markedly improve cell-seeding efficiency to nonfunctionalized polystyrene surfaces, as well as establish cell attachment and growth of human dermal microvascular endothelial cells on bare polyethylene terephthalate and polytetrafluoroethylene (PTFE) substrates. Scanning electron microscopy images revealed a uniform endothelial cell layer and cell-substratum compliance with the functionalized silk protein to PTFE surfaces. The co-seeding technique holds a great promise as a method to reliably and quickly cellularize engineered vascular constructs as well as to in vitro endothelialize commercially available cardiovascular grafts.

  • 34.
    Tegel, Hanna
    et al.
    KTH.
    Hedhammar, My
    KTH.
    Uhlén, Mathias
    KTH.
    Ottosson, J.
    KTH.
    Hober, Sophia
    KTH.
    Novel flow cytometry-based method for analysis of protein production in Escherichia coli2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 8, s. S66-S66Artikel i tidskrift (Övrigt vetenskapligt)
  • 35.
    Tegel, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO).
    Flow cytometry-based analysis of promoter effects on solubility of recombinantly expressed proteins2007Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 131, nr 2, s. S9-S9Artikel i tidskrift (Övrigt vetenskapligt)
  • 36. Thatikonda, Naresh
    et al.
    Delfani, Payam
    Jansson, Ronnie
    Petersson, Linn
    Lindberg, Diana
    Wingren, Christer
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Swedish University of Agricultural Sciences, Sweden.
    Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays2016Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 11, nr 3Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.

  • 37.
    Thatikonda, Naresh
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Nilebäck, Linnea
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Kempe, Adam
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Widhe, Mona
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in Vitro Cell Culture: A Step toward Creation of Artificial ECM2018Ingår i: ACS Biomaterials Science and Engineering, ISSN 2373-9878, Vol. 4, nr 9, s. 3384-3396Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Presentation of immobilized growth factors with retained bioactivity remains a challenge in the field of tissue engineering. In the present study, we propose a strategy to covalently conjugate a pleiotropic growth factor, basic fibroblast growth factor (bFGF) to a partial spider silk protein at gene level. The resulting silk-bFGF fusion protein has the propensity to self-assemble into silk-like fibers, and also surface coatings, as confirmed by quartz crystal microbalance studies. Functionality of the silk-bFGF coating to bind its cognate receptor was confirmed with surface plasmon resonance studies. As a step toward the creation of an artificial ECM, the silk-bFGF protein was mixed with FN-silk, an engineered spider silk protein with enhanced cell adhesive properties. Bioactivity of the thereby obtained combined silk was confirmed by successful culture of primary human endothelial cells on coatings and integrated within fibers, even in culture medium without supplemented growth factors. Together, these findings show that silk materials bioactivated with growth factors can be used for in vitro cell culture studies, and have potential as a tissue engineering scaffold.

  • 38.
    Uhlén, Mathias
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Björling, Erik
    KTH, Skolan för bioteknologi (BIO).
    Agaton, Charlotta
    KTH, Skolan för bioteknologi (BIO).
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för bioteknologi (BIO).
    Amini, Bahram
    KTH, Skolan för bioteknologi (BIO).
    Andersen, Elisabet
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Andersson, Ann-Catrin
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Angelidou, Pia
    KTH, Skolan för bioteknologi (BIO).
    Asplund, Anna
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Asplund, Caroline
    KTH, Skolan för bioteknologi (BIO).
    Berglund, Lisa
    KTH, Skolan för bioteknologi (BIO).
    Bergström, Kristina
    KTH, Skolan för bioteknologi (BIO).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO).
    Cerjan, Dijana
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Ekström, Marica
    KTH, Skolan för bioteknologi (BIO).
    Elobeid, Adila
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Eriksson, Cecilia
    KTH, Skolan för bioteknologi (BIO).
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO).
    Falk, Ronny
    KTH, Skolan för bioteknologi (BIO).
    Fall, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Forsberg, Mattias
    KTH, Skolan för bioteknologi (BIO).
    Gry Björklund, Marcus
    KTH, Skolan för bioteknologi (BIO).
    Gumbel, Kristoffer
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Halimi, Asif
    KTH, Skolan för bioteknologi (BIO).
    Hallin, Inga
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Hamsten, Carl
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Hansson, Marianne
    KTH, Skolan för bioteknologi (BIO).
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO).
    Hercules, Görel
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Kampf, Caroline
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Larsson, Karin
    KTH, Skolan för bioteknologi (BIO).
    Lindskog, Mats
    KTH, Skolan för bioteknologi (BIO).
    Lodewyckx, Wald
    KTH, Skolan för bioteknologi (BIO).
    Lund, Jan
    KTH, Skolan för bioteknologi (BIO).
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Magnusson, Kristina
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Malm, Erik
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Ödling, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO).
    Olsson, Ingmarie
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Öster, Emma
    KTH, Skolan för bioteknologi (BIO).
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO).
    Paavilainen, Linda
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rimini, Rebecca
    KTH, Skolan för bioteknologi (BIO).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO).
    Runeson, Marcus
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Sköllermo, Anna
    KTH, Skolan för bioteknologi (BIO).
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO).
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO).
    Sterky, Fredrik
    KTH, Skolan för bioteknologi (BIO).
    Strömberg, Sara
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO).
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO).
    Tourle, Samuel
    KTH, Skolan för bioteknologi (BIO).
    Wahlund, Eva
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Waldén, Annelie
    KTH, Skolan för bioteknologi (BIO).
    Wan, Jinghong
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Westberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Wester, Kenneth
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    Wrethagen, Ulla
    KTH, Skolan för bioteknologi (BIO).
    Xu, Lan Lan
    KTH, Skolan för bioteknologi (BIO).
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Pontén, Fredrik
    Uppsala Univ, Rudbeck Lab, Dept Genet & Pathol.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 12, s. 1920-1932Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

  • 39. Widhe, M.
    et al.
    Bysell, H.
    Nystedt, S.
    Schenning, I.
    Malmsten, M.
    Johansson, J.
    Rising, A.
    Hedhammar, My
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Biomed Ctr, S-75123 Uppsala, Sweden.
    Recombinant spider silk as matrices for cell culture2010Ingår i: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 31, nr 36, s. 9575-85Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The recombinant miniature spider silk protein, 4RepCT, was used to fabricate film, foam, fiber and mesh matrices of different dimensionality, microstructure and nanotopography. These matrices were evaluated regarding their suitability for cell culturing. Human primary fibroblasts attached to and grew well on all matrix types, also in the absence of serum proteins or other animal-derived additives. The highest cell counts were obtained on matrices combining film and fiber/mesh. The cells showed an elongated shape that followed the structure of the matrices and exhibited prominent actin filaments. Moreover, the fibroblasts produced, secreted and deposited collagen type I onto the matrices. These results, together with findings of the matrices being mechanically robust, hold promise not only for in vitro cell culturing, but also for tissue engineering applications.

  • 40.
    Widhe, Mona
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Shalaly, Nancy Dekki
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    A fibronectin mimetic motif improves integrin mediated cell biding to recombinant spider silk matrices2016Ingår i: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 74, s. 256-266Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cell binding motif RGD is the most widely used peptide to improve cell binding properties of various biomaterials, including recombinant spider silk. In this paper we use genetic engineering to further enhance the cell supportive capacity of spider silk by presenting the RGD motif as a turn loop, similar to the one found in fibronectin (FN), but in the silk stabilized by cysteines, and therefore denoted FNCC. Human primary cells cultured on FNCC-silk showed increased attachment, spreading, stress fiber formation and focal adhesions, not only compared to RGD-silk, but also to silk fused with linear controls of the RGD containing motif from fibronectin. Cell binding to FNCC-silk was shown to involve the alpha 5 beta 1 integrin, and to support proliferation and migration of keratinocytes. The FNCC-silk protein allowed efficient assembly, and could even be transformed into free standing films, on which keratinocytes could readily form a monolayer culture. The results hold promise for future applications within tissue engineering.

  • 41. Yang, L.
    et al.
    Hedhammar, My
    Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 575, Uppsala.
    Blom, T.
    Leifer, K.
    Johansson, J.
    Habibovic, P.
    van Blitterswijk, C. A.
    Biomimetic calcium phosphate coatings on recombinant spider silk fibres2010Ingår i: Biomedical Materials, ISSN 1748-6041, E-ISSN 1748-605X, Vol. 5, nr 4, artikel-id 045002Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant minispidroins. In the present study, supersaturated simulated body fluid was used to deposit calcium phosphate coatings on recombinant spider silk fibres. The mineralization process was followed in time using scanning electron microscopy equipped with an energy dispersive x-ray (EDX) detector and Raman spectroscope. Focused ion beam technology was used to produce a cross section of a coated fibre, which was further analysed by EDX. Preliminary in vitro experiments using a culture of bone marrow-derived human mesenchymal stem cells (hMSCs) on coated fibres were also performed. This study showed that recombinant spider silk fibres were successfully coated with a homogeneous and thick crystalline calcium phosphate layer. In the course of the mineralization process from modified simulated body fluid, sodium chloride crystals were first deposited on the silk surface, followed by the deposition of a calcium phosphate layer. The coated silk fibres supported the attachment and growth of hMSCs.

1 - 41 av 41
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