• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.184-190
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• 2007

Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.196-205
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• 2015

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1634-1640
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• 2008

This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1243-1250
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• 2010

A cDNA encoding a cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum, and the ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 kDa as deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5${\alpha}$ harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5${\alpha}$ and control.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1362-1368
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• 2006

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

• Reproductive and Developmental Biology
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• v.40 no.1
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• pp.7-13
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• 2016

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat ${\beta}$-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.260-267
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• 2008

The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system (pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and $\beta$-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of $2{\sim}3%$ of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.49-55
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• 2005

We cloned cDNA of the PGH(porcine growth hormone) gene and constructed retrovirus vector designed to express PGH gene under the regulation of CMV (cytomegalovirus) promoter. To maximize the expression, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was placed at the downstream of the PGH gene. After infection with recombinant viruses, approximately 1×10/sup 6/ PFF(porcine fetal fibroblast) cells released PGH protein into the media as much as 1,400 ng. In a subsequent experiment, a modifications of the retrovirus vector was made to express the PGH gene in a teracycline-inducible manner. In PFF cells carrying these viral vector sequences, addition of doxycycline to the media resulted in 2∼6 fold increase in PGH synthesis. In the modified retrovirus vectors, the WPRE sequence also played a role in boosting the effect of the tetracycline induction. This result indicates that our tetracycline-inducible expression system might be a promising candidate in alleviating the complicate physiological problems caused by constitutive expression of the exogenous genes in the transgenic animals.

• Journal of Industrial Technology
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• v.30 no.B
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• pp.69-74
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• 2010

Chitinolytic activity was enhanced by coexpression of endo-chitinase gene (chiA) and chitobiosidase gene (chiB) from Serratia marcescens KFRI314 using constitutive expression vector, pHCEIA, in E. coli. Coexpression vector was constructed by inserting ribosome binding site (RBS) into junction between two chitinase genes. SDS-PAGE analyses showed that two chitinase were constitutively expressed while E. coli clones expressing two chitinases simultaneously increased halo size on colloidal chitin plate. Furthermore, the chitinolytic activities were much enhanced in coexpressed clones when degradation patterns of substrate analogues such as 4-MU-(NAG), $4-MU-(NAG)_2$,$4-MU-(NAG)_3$ were used. Consequently, the combined use of endochitinase and chitobiosidase greatly increased overall chitinolytic activities on recombinant E. coli clones.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.57-62
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• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.