• Biomolecules & Therapeutics
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• v.3 no.4
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• pp.311-315
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• 1995

The K. aerogenes ureal gene product was previously shown to facilitate assembly of the crease metallocenter (Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y., and Hausinger, R. P. (1992) J. Bacteriol. 174, 4324-4330). UreG protein has now been purified and characterized. Although the protein is predicted to possess a putative NTP-binding P-loop motif, equilibrium dialysis studies showed negative results. Immunogold electron microscopic studies using polyclonal antibodies directed against UreG protein confirm that UreG is located in the cytoplasm as predicted in the DNA sequence.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.274-279
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• 2015

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-_D-1-thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

• Archives of Pharmacal Research
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• v.29 no.5
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• Journal of Microbiology
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• v.37 no.1
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• pp.21-26
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• 1999

Two novel calcium-binding proteins, named CAB-I and CAB-II, have been isolated from Streptomyces coelicolor. Purification of the calcium-binding proteins involved heat treatment, fractionation with ammonium sulfate, acid treatment, anion exchange and hydrophobic interaction column chromatography, FPLC gel filtration, and preparative isoelectric focusing. A chelex competitive assay and 45Ca autoradiography verified the calcium-binding ability of the proteins. The major band CAB-II has an apparent molecular weight of 26,000 determined by SDS-polyacrylamide gel electrophoresis and 340,000 determined by gel filtration. The isoelectric point of this molecule showed the acidic nature of the molecule. N-terminal amino acid sequence analysis shows homology to rat Ca2+/calmodulin-dependent protein kinase-II (CAB-II) and yeast phosphoprotein phosphatase (CAB-I).

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1002-1010
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• 2001

The D-xylose operon in Escherichia coli is known to be regulated by a transcriptional activator protein, XyIR, which is responsible for the expression of both xylAB and xylFGH gene clusters. The XyIR was purified to homogeneity by using the maltose binding protein fusion expression and purification systems involving two chromatography steps. The purified XyIR protein was composed of two subunits of 45 kDa, which was determined by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The purified XyIR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR to the xylA promoter was enhanced by adding xylose. The enhanced binding ability of XyIR in the presence of xylose was not diminished by adding glucose. The presumed XyIR binding site is located between 120 bp to 100 bp upstream the xylA initiation codon.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• Journal of Plant Biology
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• v.39 no.3
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• pp.173-177
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• 1996

A novel calcium binding protein (CaBP) was purified to electrophoretic homogeneity from Dunaliella salina. In the course of purification experiment, this CaBP was identified as a monomer and its molecular weight was about 21 kDand isoelectric point (pI) value was about 4.1 using isoelectrofocusing. This CaBP was able to bind Ca2+ even in the pressence of an excess MgCl2 and KCI both in solution. In the SDS-PAGE, the Ca2+-bound form was slower than the Ca2+-free form in the nondenaturing PAGE. This means that the CaBP undergoes conformational change in the Ca2+-bound condition. Furthermore, UV absorption spectrum and fluorescence intensity of this CaBP was investigated. UV absorption peak was appeared at about 258 nm and decreased somewhat in Ca2+-bound condition. In the measurement of fluorescence, maximum intensity was appeared at 303 nm and decreased in Ca2+-bound state, similarly as UV absorption spectrum. These show distinct changes upon Ca2+-binding, which indicate of structural and/or dynamic changes largely reminiscent of other members of the EF-hand Ca2+-binding protein family.

• The Korean Journal of Mycology
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• v.11 no.1
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• pp.23-26
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• 1983

Aspergillus ustus was cultured in the salts media contained dextran (2%). Then the cultured liquid media were filtrated and concentrated up to 10 folds by evaporation, and then purified by means of acetone precipitation, of a repeated chromatography on the columns of DEAE-Ccellulose, Biogel P-150, and Sephadex G-200. Total proteins in the initial culture filtrate were 38,500mg, but the final amounts of proteins were 172mg. The specific activity of the protein in the culture filtrate was $1,340\;{\mu}moles$ products per minute per mg protein, but the final specific activity of the protein was $2,448\;{\mu}\;moles$ products per minute per mg protein. The final yields remained about 30% of the initial.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.122-127
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• 2005

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human $interferon-\gamma$ ($rhIFN-\gamma$) at a high concentration. The $rhlFN-\gamma$ was overexpressed in E. coli resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the buffer-exchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded $rhIFN-\gamma$, with protein recovery of $67.1\%$ and specific activity up to $1.2\times10^7\;IU/mg$.