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  • 1. Ehn, M.
    et al.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein2001In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 22, no 1, p. 120-127Article in journal (Refereed)
    Abstract [en]

    Escherichia coli (E. coli) single-stranded binding protein (SSB) is a valuable protein for various biotechnical applications, such as PCR and DNA sequencing, Here we describe an efficient expression and purification scheme where the tendency of SSB to aggregate at low salt concentration and high protein concentration is avoided. The method contains fewer steps of purification and results in high protein yield, compared to previous published protocols. In our protocol, cells are harvested after cultivation overnight and SSB is isolated by ammonium sulfate precipitation followed by anion-exchange chromatography. The yield from a 2-liter fed-batch fermenter is 2 g protein, which is higher than all production methods for SSB earlier reported, Moreover, the two classical isolation steps combined in the purification scheme are robust, cost-efficient, and suitable for scaling up. The resulting SSB is pure and a correctly folded tetramer with an apparent binding to single-stranded DNA with a K-D of 10(-8) M, as determined by surface plasmon resonance.

  • 2.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nilsson, Joakim
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lindberg, Michael
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein1997In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, p. 125-132Article in journal (Refereed)
    Abstract [en]

    A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.

  • 3.
    Hedhammar, My
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Jung, H. R.
    University of Southern Denmark, Department for Biochemistry and Molecular Biology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Enzymatic cleavage of fusion proteins using immobilised protease 3C2006In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 47, no 2, p. 422-426Article in journal (Refereed)
    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.

  • 4.
    Kürten, Charlotte
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Overexpression of functional human oxidosqualene cyclase in Escherichia coli2015In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 115, p. 46-53Article in journal (Refereed)
    Abstract [en]

    The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7 mg protein/g cells (hOSC in Pichia pastoris) after 48 h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9 mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.

  • 5. Molina, Daniel Martinez
    et al.
    Lundbäck, Anna-Karin
    KTH, School of Technology and Health (STH).
    Niegowski, Damian
    Eshaghi, Said
    Expression and purification of the recombinant membrane protein YidC: A case study for increased stability and solubility2008In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 62, no 1, p. 49-52Article in journal (Refereed)
    Abstract [en]

    YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, translocation and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 mu g of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high concentrations and that was stable for weeks at +4 degrees C. The isolated protein is thus well suited for structural studies.

  • 6. Nourizad, N.
    et al.
    Ehn, M.
    Gharizadeh, B.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)2003In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 27, no 2, p. 229-237Article in journal (Refereed)
    Abstract [en]

    ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1 mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48 kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

  • 7. Rotticci-Mulder, J. C.
    et al.
    Gustavsson, M.
    Holmquist, M.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Martinelle, Mats
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain2001In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 21, no 3, p. 386-392Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the a-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.

  • 8. Sjodahl, J.
    et al.
    Emmer, Åsa
    KTH, Superseded Departments, Chemistry.
    Vincent, J.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Characterization of proteinases from Antarctic krill (Euphausia superba)2002In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 26, no 1, p. 153-161Article in journal (Refereed)
    Abstract [en]

    Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes. CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060. and 26,260 Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260 Da. whereas the CPB enzyme masses likely are 33,730 and 33,900 Da, The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degreesC and 1-3 degreesC, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.

  • 9.
    Steen, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    High-throughput protein purification using an automated set-up for high-yield affinity chromatography2006In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 46, no 2, p. 173-178Article in journal (Refereed)
    Abstract [en]

    One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5 h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.

  • 10.
    Tegel, Hanna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Tourle, Samuel
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)2010In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 69, no 2, p. 159-167Article in journal (Refereed)
    Abstract [en]

    The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).

  • 11.
    Wittrup Larsen, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, Uwe T.
    Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems2008In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 62, p. 90-97Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CALB) carrying a point mutation, N74S, resulting in a non-glycosylated protein was actively expressed in Pichia pastoris yielding 44 mg/L which was similar to that of the glycosylated CALB wild type expressed in A pastoris. Hence, the major obstacle in the Escherichia coli expression of CALB is not the lack of glycosylation. To understand and improve the expression of CALB in E. coli, a comprehensive investigation of four different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3) and Origami 2(DE3) as well as co-expression with chaperones groES and groEL in Origami B(DE3), all using the pET-22b(+) vector and the T7lac promoter.

    Furthermore the E. coli expression was carried out at three different temperatures (16, 25 and 37 C) to optimise the expression. Periplasmic expression resulted in highest amount of active CALB of the four systems, yielding a maximum of 5.2 mg/L culture at 16 degrees C, which is an improvement to previous reports.

    The specific activity of CALB towards tributyrin in E. coli was found to be the same for periplasmic and cytosolic expression. Active site titration showed that the CALB mutant N74S had a lower specific activity in comparison to wild type CALB regardless of expression host. The expected protein identity was confirmed by LC-ESI-MS analysis in E. coli, whereas in P. pastoris produced CALB carried four additional amino acids from an incomplete protein processing.

  • 12. Wållberg, Helena
    et al.
    Löfdahl, Per-Åke
    Tschapalda, Kirsten
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Tolmachev, Vladimir
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand2011In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 76, no 1, p. 127-135Article in journal (Refereed)
    Abstract [en]

    Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.

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