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  • 1.
    Boström, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Markland, Katrin
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Sandén, Anna Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli2005In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, no 1, p. 82-90Article in journal (Refereed)
    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.
    Guo, Weijin
    et al.
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Gustafsson, Linnea
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    van der Wijngaart, Wouter
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Formation of a thin-walled Spider Silk Tube on a Micromachined Scaffold2018In: Proceeding of 2018 IEEE 31st International Conference on Micro Electro Mechanical Systems (MEMS), Institute of Electrical and Electronics Engineers (IEEE), 2018, Vol. 2018, p. 83-85Conference paper (Refereed)
    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.

  • 3.
    Kanje, Sara
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Venskutonytė, Raminta
    Lund University.
    Scheffel, Julia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Nilvebrant, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Lindkvist-Petersson, Karin
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Protein engineering allows for mild affinity-based elution of therapeutic antibodies2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, p. 3427-3438Article in journal (Refereed)
    Abstract [en]

    Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification

  • 4.
    Lindbo, Sarah
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Garousi, Javad
    Uppsala university.
    Mitran, Bogdan
    Uppsala university.
    Vorobyeva, Anzhelika
    Uppsala university.
    Oroujeni, Maryam
    Uppsala university.
    Orlova, Anna
    Uppsala university.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Tolmachev, Vladimir
    Uppsala university.
    Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 7, p. 2674-2683Article in journal (Refereed)
    Abstract [en]

    Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.

  • 5.
    Nilebäck, Linnea
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hedin, Jesper
    Widhe, Mona
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Floderus, Lotta S.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Krona, Annika
    Bysell, Helena
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study2017In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 18, no 3, p. 846-854Article in journal (Refereed)
    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.

  • 6.
    Råvik, Mattias
    et al.
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Cimander, Christian
    Elofsson, Ulla
    Veide, Andres
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    A method for microbial cell surface fingerprinting based on surface plasmon resonance2007In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 70, no 4, p. 595-604Article in journal (Refereed)
    Abstract [en]

    A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).

  • 7.
    Sandén, Anna Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Impact of glucose feed rate on productivity and recombinant protein quality in Escherichia coli2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate.

    The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, qp, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins.

    By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation,

    In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied.

    In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the PmalK production system.

    A novel fed-batch process based on the promoters for the universal stress proteins A and B (PuspA, PuspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects.

  • 8.
    Sandén, Anna Maria
    et al.
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Boström, Maria
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Markland, Katrin
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli2005In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 2, p. 239-247Article in journal (Refereed)
    Abstract [en]

    From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation.

    Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure.

    It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (<= 5 mu M), this formation is almost abolished while at a comparatively high concentration (>= 300 mu M) 50% of the total product accumulated was in the form of inclusion bodies.

  • 9.
    Wu, Yu-Tang
    et al.
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Qiu, Xue
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Susumu, Kimihiro
    US Naval Res Lab, Opt Sci Div, Code 5600, Washington, DC USA.;KeyW Corp, Hanover, MD 21076 USA..
    Medintz, Igor L.
    US Naval Res Lab, Ctr Bio Mol Sci & Engn, Code 6900, Washington, DC USA..
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hildebrandt, Niko
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins2018In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 14, no 35, article id 1802266Article in journal (Refereed)
    Abstract [en]

    Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Forster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (approximate to 6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab', or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 x 10(-12)m (approximate to 8 ng mL(-1)) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.

  • 10.
    Zhang, Ye
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Stobbe, Per
    Silvander, Christian Orrego
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor2015In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 213, p. 28-41Article in journal (Refereed)
    Abstract [en]

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 x 106 viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 C to 29 C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

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