• KSBB Journal
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• v.23 no.5
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• pp.445-448
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• 2008

Candida antarctica lipase B (CalB) is an efficient biocatalyst for many organic synthesis reactions. To make full use of CalB, we need effective expression system. Previously recombinant CalB was successfully expressed in the methylotropic yeast Pichia pastoris. In addition, we succeed in the functional expression of CalB in the Escherichia coli cytoplasm. This CalB expression system in E.coli has many considerable advantages in comparison with other expression systems and enables high-throughput screening of gene libraries as those derived from directed evolution experiments. To optimize E.coli system, we investigate comparing between OrigamiB (DE3) and BL21 (DE3) and observing effect of IPTG amount.

• KSBB Journal
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• v.23 no.5
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• pp.403-407
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• 2008

Recently, Candida antarctica lipase B (CalB) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, CalB, which is stable under harsh condition, is in demand from many industries. In order to reform CalB promptly, the expression system which has advantages of ease to use and low cost for gene libraries screening was developed using E. coli. The E. coli strains, Rosettagami with competence for enhanced disulfide bond formation, Novablue, and $DH5{\alpha}$, were exploited in this study. To obtain the soluble CalB, the pCold I vector expressing the cloned gene at $15^{\circ}C$ and the chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL were used for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected by employing the halo plate containing 1% tributyrin, and the CalB expression was confirmed by SDS-PAGE. E. coli Rosettagami and $DH5{\alpha}$ harbouring groES/groEL chaperones were able to express soluble CalB effectively. From a facilitative point of view, E. coli $DH5{\alpha}$ is more suitable for further mutation study.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• IMMUNE NETWORK
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• v.10 no.2
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• pp.35-45
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• 2010

Background: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. Methods: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize $V_H$ and $V_L$ fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. Results: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of $V_H$ or $V_L$ domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of VH of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. Conclusion: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

• KSBB Journal
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• v.24 no.4
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• pp.341-346
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• 2009

Candida Antarctica lipase A (CalA) has been used because of its suitability in industrial applications. CalA has unique features capable to accept tertiary and sterically hindered alcohols among many hydrolases. CalA gene was cloned and constructed in expression vector such as pColdIII/CalA and $pPICZ{\alpha}A$/CalA. The gene encoding pColdIII/CalA was functionally expressed in the cytoplasm of Escherichia coli $Origami^{TM}$ B (DE3) cells. The plasmid $pPICZ{\alpha}A$/CalA linearized by BstX I was integrated into 5'AOX1 region of the chromosomal DNA and was functionally expressed in the methyl atrophic yeast Pichia pastoris. Expressed CalA in P. pastoris (0.7 Unit/mL) showed 35 times higher activity than that in E. coli expression system (0.02 Unit/mL).