• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.660-665
• /
• 2003

High-level expression of Thermus caldophilus GK24 alkaline phosphatase (Tca APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAP1 and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tca APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAPl. A rapid purification procedure of Tca APase overproduced in E. coli was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and $Mg^{2+}$ dependence of the recombinant Tca APase were similar to those of native enzyme isolated from T. caldophilus GK24.

• Journal of Life Science
• /
• v.25 no.6
• /
• pp.631-637
• /
• 2015

Streptavidin, a protein produced by Streptomyces avidinii, strongly binds up to four molecules of vitamin H, d-biotin exhibiting the dissociation constant of about 10⁻¹⁵ M. This strong binding affinity has been applied for detection and characterization of numerous biological molecules suggesting expression and purification of functional streptavidin should be very useful for the application of this streptavidin-biotin interaction. To express a soluble streptavidin in Escherichia coli, We synthesized streptavidin genes and cloned into pET-22b plasmid, which uses T7 RNA polymerase/T7 promoter expression systems containing pelB leader for secretion into periplasmic space and six polyhistidine tags at C-terminus for purification of expressed proteins. Although streptavidin is toxic to Escherichia coli due to strong biotin binding property, streptavidin was expressed very sufficiently in a range of 10-20 mg/ml. In SDS-PAGE, the size of purified protein was shown as 17 kDa in denatured condition (boiling) and 68 kDa in native condition (without boiling) suggesting tetramerization of monomeric subunit by non-covalent association. Further analysis by size-exclusion chromatography supported streptavidin’s tetrameric structure as well. In addition, soluble streptavidin detected biotinylated proteins in westernblot indicating its functional activity to biotin. Taken these results together, it concluded that our simple expression system was able to show high yield, homotetrameric formation and biotin binding activity analogous to natural streptavidin.

• Microbiology and Biotechnology Letters
• /
• v.26 no.1
• /
• pp.15-22
• /
• 1998

The dnrF gene, responsible for conversion of aklavinone to $\varepsilon$-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin gene cluster of Streptomyces peucetius subsp. caesius ATCC 27952, close to drrAB, one of the anthracycline resistance genes. To characterize the enzymatic properties of the aklavinone 11-hydroxylase, the dnrF gene was overexpressed in Escherchia coli. The pET-22(+) plasmid which has the T7 promoter under the control of lacUV5 gene was used for the overexpression of the dnrF gene, and the recombinant plasmid pET213 that contains the dnrF gene linked to the T7 promoter of pET-22b(+) was introduced into the E. coli BL2l. When the expression of the dnrF gene was induced by IPTG at the final concentration of 1 mM, the induced protein could be detected in SDS-PAGE only in insoluble precipitate. The insoluble protein was electroeluted from the gel and used for the preparation of antiserum in mice. Various culture conditions were tested to maximize the expression of the aklavinone 11-hydroxylase in soluble form. The enzymatic activity was checked by the bioconversion experiment, and the protein was confirmed by the SDS-PAGE and the Western blot analysis. From the analysis of the data, it was concluded that the culture induced with IPTG at the final concentration of 0.02 mM at 37$^{\circ}C$ yielded the best productivity of active form of enzyme.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.5
• /
• pp.612-619
• /
• 2000

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.