• KSBB Journal
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• v.21 no.2
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• pp.118-122
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• 2006

In this report, we describe that the use of GroEL/GroES-enriched S30 extract remarkably enhances the solubility and enzymatic activity of cell-free synthesized rPA, which requires the correct formation of 9 disulfide bonds for its biological activity. We found that the stable maintenance of redox potential is necessary, but not sufficient for the optimal expression of active rPA. In a control reaction without using additional molecular chaperones, most of the rPA molecules were aggregated almost instantly after their expression and thus failed to exhibit the enzymatic activity. However, by the use of GroEL/GroES-enriched extract, combined with IAM-treatment, approximately $30{\mu}g/ml$ of active rPA was expressed in the cell-free synthesis reaction. This result not only demonstrates the efficient production of complex proteins, but also shows the control and flexibility offered by the cell-free protein synthesis system.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.414-419
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• 2006

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasm ids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

• Microbiology and Biotechnology Letters
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• v.43 no.3
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• pp.212-218
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• 2015

Fucosyltransferases (FucTs) catalyze fucosyl transfer from guanosine-diphosphate fucose (GDP-β-L-fucose) to acceptor molecules to form fucosyloligosaccharides with α-glycosidic linkages. However, when FucT genes have been expressed in Escherichia coli, most cases have resulted in the production of inclusion bodies. In this study, to overcome this drawback, molecular chaperones were co-expressed with α1,2-fucosyltransferase (FucT2) in E. coli. For this, the pACYC184 vector, having genes for chaperones such as GroEL, GroES, DnaK, DnaJ, and GrpE, were transformed into E. coli BL21 (DE3) star harboring pHFucT2, including the FucT2 gene from Helicobacter pylori 26695. The results from SDS-PAGE showed that 5 chaperones were successfully expressed and the soluble fraction of FucT2 was also increased. HPLC analysis revealed that the coexpression of chaperone proteins resulted in a 5-fold increase in the total activity of fucosyltransferase in E. coli. In conclusion, the FucT2 expression system developed in this study can be used as a useful tool for the synthesis of fucosyloligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.228-233
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• 2008

Effects of chaperones on mRNA stability and gene expression were studied in order to develop an efficient Escherichia coli expression system that can maximize gene expression. The stability of mRNA was modulated by introducing various secondary structures at the 5'-end of mRNA. Four vector systems providing different 5'-end structures were constructed, and genes encoding GFPuv and endoxylanase were cloned into the four vector systems. Primer extension assay revealed different mRNA half-lives depending on the 5'-end secondary structures of mRNA. In addition to the stem-loop structure at the 5'-end of mRNA, coexpression of dnaK-dnaJ-grpE or groEL-groES, representative heat-shock genes in E. coli, increased the mRNA stability and the level of gene expression further, even though the degree of stabilization was varied. Our work suggests that some of the heat-shock proteins can function as mRNA stabilizers as well s protein chaperones.

• Microbiology and Biotechnology Letters
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• v.42 no.2
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• pp.190-193
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• 2014

CMP-Neu5Ac synthetase is a key enzyme for the synthesis of CMP-Neu5Ac, which is an essential precursor of sialylated glycoconjugates. For the soluble expression of the CMP-Neu5Ac synthetase gene (neuA) from Escherichia coli K1, various heat shock proteins were co-expressed in E. coli BL21 (DE3) Star. In order to do this, a pG-KJE8 plasmid, encoding genes for GroEL-ES and DnaK-DnaJ-GrpE, was co-transformed with neuA and was expressed at $20^{\circ}C$ by the addition of 0.01 mM IPTG and 0.005 mg/ml L-arabinose. The co-expression of a variety of heat shock proteins resulted in the remarkably improved production of soluble CMP-Neu5Ac synthetase in E. coli.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.299-307
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• 2008

Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by $Ni^{2+}$ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.