| 1. |
- Bergman, Anna Carin, et al.
(author)
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dUTPase from the Retrovirus Equine Infectious Anemia Virus: High-Level Expression in Escherichia coli and Purification
- 1995
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 6:3, s. 379-387
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Journal article (peer-reviewed)abstract
- Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21(DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein, This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter−1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.8 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.
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| 2. |
- Berti, Paul J, et al.
(author)
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Affinity purification and elimination of methionine oxidation in recombinant human cystatin C
- 1997
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 11:1, s. 111-118
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Journal article (peer-reviewed)abstract
- Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.
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| 3. |
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| 4. |
- Bratt, T, et al.
(author)
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Expression of rat alpha 1-microglobulin-bikunin in baculovirus-transformed insect cells
- 1995
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 6:4, s. 8-431
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Journal article (peer-reviewed)abstract
- cDNA encoding rat alpha 1-microglobulin-bikunin was ligated into the transfer vector pVL 1392 and recombined with a wild-type baculovirus. The resulting alpha 1-microglobulin-bikunin-encoding baculovirus was used to infect Trichoplusia ni (Hi-5) insect cells. The infected cells secreted alpha 1-microglobulin with maximal concentrations of 15 mg/liter 5 days after infection. The secreted proteins migrated upon SDS-PAGE as two major protein bands, 40 and 26 kDa, corresponding to alpha 1-microglobulin-bikunin and free alpha 1-microglobulin. The results suggested that the cells secreted mostly alpha 1-microglobulin-bikunin, which subsequently was cleaved in the medium, yielding free alpha 1-microglobulin. Both forms were isolated by monoclonal anti-alpha 1-microglobulin affinity chromatography, and alpha 1-microglobulin-bikunin separated from free alpha 1-microglobulin by gel chromatography. The yields of purified alpha 1-microglobulin-bikunin and free alpha 1-microglobulin were approximately 1 and 5 mg, respectively, per liter medium. Insect cell alpha 1-microglobulin displayed a size, shape, and charge heterogeneity similar to alpha 1-microglobulin isolated from rat urine. A panel of monoclonal antibodies raised against urinary alpha 1-microglobulin from several different species bound to rat urinary alpha 1-microglobulin and insect cell secreted alpha 1-microglobulin-bikunin and free alpha 1-microglobulin with approximately the same strength, indicating that the three proteins are folded in similar ways. The results of glycosidase treatments and lectin blotting indicate the absence of neuraminic acid but the presence of one N-linked oligosaccharide and an unspecified number of O-linked oligosaccharides in alpha 1-microglobulin-bikunin and free alpha 1-microglobulin.
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| 5. |
- Ejdebäck, Mikael, 1969-, et al.
(author)
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Effects of codon usage and vector-host combinations on the expression of spinach plastocyanin in Escherichia coli
- 1997
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In: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 11:1, s. 17-25
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Journal article (peer-reviewed)abstract
- Spinach plastocyanin has been expressed in Escherichia coli and exported to the periplasmic space. The effects of codon usage, expression system, growth length, and temperature on expression levels in LB medium were investigated. A stretch of codons, rare in E. coli, was identified and replaced with highly expressed codons, increasing the yield by at least 20%. Plastocyanin was more efficiently expressed under the T7 promoter than under the lac promoter. Maximum yields were obtained at 37 degrees C when growing the cells for 16 h after induction. The optimized expression system produced 38 mg holoprotein per liter culture. In this system it was also possible to express plastocyanin in minimal medium, at a yield of 10 mg per liter. N-terminal sequencing and mass spectrometry showed that plastocyanin was correctly processed. The expressed plastocyanin was purified to homogeneity, as shown by an A278/A597 ratio of 1.0, and together with amino acid analysis and the determination of oxidized and total copper contents, both the absorption coefficients for epsilon 278 and for epsilon 597 were determined to be 4700 M-1 cm-1.
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| 6. |
- Gräslund, Torbjörn, et al.
(author)
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Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
- 1997
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 9, s. 125-132
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Härndahl, Ulrika, et al.
(author)
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The Chloroplast Small Heat Shock Protein-Purification and Characterization of Pea Recombinant Protein
- 1998
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 14:1, s. 87-96
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Journal article (peer-reviewed)abstract
- We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using anEscherichia coliexpression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5.0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment. This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens @a-crystallin. The crystallins are known from both crystallographic and spectroscopic data to be all-@b proteins. In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% @a-helical secondary structure.
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