• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.451-456
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• 2003

An expression vector system was developed for the secretory production of recombinant Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae. The mature L1 lipase gene was fused to ${\alpha}-amylase$ signal sequence from Aspergillus oryzae for the effective secretion into the culture broth and the expression was controlled under GAL10 (the gene coding UDP-galactose epimerase of S. cerevisiae) promoter. S. cerevisiae harboring the resulting plasmid successfully secreted L1 lipase into the culture broth. To examine an optimum condition for L1 lipase expression in the fed-batch culture, L1 lipase expression was induced at three different growth phases (early, mid, and late-exponential growth phases). Maximum product on of L1 lipase (1,254,000 U/l, corresponding to 0.65/1) was found when the culture was induced at an early growth phase. Secreted recombinant L1 lipase was purified only through CM-Sepharose chromatography, and the purified enzyme showed 1,963 U/mg of specific activity and thermoalkalophilic properties similar to those reported for the enzyme expressed in Escherichia coli.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.6
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• pp.743-749
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• 2004

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.640-646
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• 1990

We have cloned the cdd gene from E. coli C600 using (cdd-) as a host. From the sequenced promoter region of E=, coli cdd gene which has been determined by Valentin-Hansen P. (1985), we synthesized the 23 mer oligonucleotides corresponding to the transcription initiation region and used as a probe for cloning of the cdd gene by Southern blotting. The isolated fragments in the blotting were introduced to the colony hybridization after transforming it into the E. coli JF611 (cdd-, pyr double mutant), and we identified the hybridized band at 27 kb long. From the original insert of 27 kb fragment in theBamHI site of pBR322, the 5.3 kb fragment containing the cdd gene was isolated by subsequent deletion and subeloning. From the derived plasmid pTK509, further deletion and subcloning were performed and clarified that the cdd gene was located in the 2.1 kb of SaZI/DraI segment in the insert of pTK605. The polypeptide encoded by the cloned DNA was appeared to be a molecular mass of 33,000.

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

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.850-855
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• 2007

Leptin is the adipocyte-specific product of the obese gene and plays a major role in food intake and energy metabolism. Leptin research was mainly focused on mammalian species, but understanding of leptin and its function in poultry is very poor. In this study, the duck leptin gene was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from duck liver RNA. The cDNA fragment was inserted into the pET-28a expression vector, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3). Experimental mice were given an intraperitoneal injection of 10 mg/kg leptin dissolved in phosphate buffered saline (PBS), while the control mice were injected with PBS. The effect of leptin on food intake, body weight and fatty deposition in mice was detected. Sequence analysis revealed that duck leptin had a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The sequence shares highly homology to other animals. The coding sequence of duck leptin was 84 and 86% identical to human and pig leptin nucleotides sequence. Highest identity was with the rat coding sequence (95%). The identity of the amino acid sequence was 84, 82 and 96% respectively compared to that of the human, pig and rat. Results of SDS-PAGE analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The purified product was found to be biologically active during tests. Continuous administration of recombinant duck leptin inhibited food intake. Despite the decrease of food intake, leptin significantly induced body weight and fatty deposition. These changes were accompanied by a significant down-secretion of plasma glucose, cholesterol, triglyceride and insulin levels in mice. The observations provide evidence for an inhibitory effect of leptin in the regulation of food intake and for a potential role of duck leptin in the regulation of lipogenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2555-2559
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• 2015

Purpose: To investigate effects of the TESTIN (TES) gene on proliferation and migration of highly metastatic nasopharyngeal carcinoma cell line 5-8F and the related mechanisms. Materials and Methods: The target gene of human nasopharyngeal carcinoma cell line 5-8F was amplified by PCR and cloned into the empty plasmid pEGFP-N1 to construct a eukaryotic expression vector pEGFP-N1-TES. This was then transfected into 5-8F cells. MTT assays, flow cytometry and scratch wound tests were used to detect the proliferation and migration of transfected 5-8F cells. Results: A cell model with stable and high expression of TES gene was successfully established. MTT assays showed that the OD value of 5-8F/TES cells was markedly lower than that of 5-8F/GFP cells and 5-8F cells (p<0.05). Flow cytometry showed that the apoptosis rate of 5-8F/TES cells was prominently increased compared with 5-8F/GFP cells and 5-8F cells (p<0.05). In vitro scratch wound assays showed that, the width of the wound area of 5-8F/TES cells narrowed slightly, while the width of the wound area of 5-8F/ GFP cells and 5-8F cells narrowed sharply, suggesting that the TES overexpression could inhibit the migration ability. Conclusions: TES gene expression remarkably inhibits the proliferation of human nasopharyngeal carcinoma cell line 5-8F and reduces its migration in vitro. Thus, it may be a potential tumor suppressor gene for nasopharyngeal carcinoma.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7523-7527
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• 2013

Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. In order to obtain pure protein for analysis of structure and biological function, an expression vector TS-pET28b (+) was constructed by inserting wild-type human thymidylate synthase (hTS) cDNA into pET28b (+). Then an expression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion protein with His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content was approximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodies were washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. On SDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecular mass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.281-286
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• 2010

A Pseudomonas fluorescens strain was isolated and found to show antagonistic activity against phytopathogenic fungi and to possess a gene responsible for production of antibiotic 2,4-diacetylphloroglucinol. For the extension of biocontrol range, a gene for an Androetonus australis Hector insect toxin 1 (AaHIT1), one of the most known toxic insect-selective peptides, was designed and synthesized according to the preferred codon usage of Pseudomonas fluorescens, cloned, and transformed into the strain by pSUP106 vector, a broad-host-range plasmid. Bioassays indicated that the engineered strain was able to produce AaHIT1 with insecticidal activity, and at the same time retain the activity against plant pathogen. The experiments for nonplanted soil and rhizosphere colonization showed that, similar to the population of the wild-type strain, that of the engineered strain remained relatively constant in the first 10 days, and the subsequent 50 days, suggesting that AaHIT1 expression in the bacterial cell does not substantially impair its long-term colonization. It is first reported that a Pseudomonas fluorescens strain expressing an active scorpion neurotoxin has dual activity against phytopathogenic fungi and insects, making at attractive for agronomic applications.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.495-499
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• 2016

In order to identify the biochemical characteristics of LuxG, the luxG gene from bioluminescence bacteria of Photobacterium leiognathi ATCC 25521 was isolated by PCR-Amplification and inserted into pQE30 vector containing the T5 promoter and 6X His-tag system. The resulting recombinant plasmid was transformed into Escherichia coli to over-express the luxG gene and purify the gene product. The purified LuxG protein demonstrated ferric iron reductase activity and the kinetic parameters of $K_m$ and $V_{max}$ for FMN as well as the NADPH substrates of ferric iron reductase were determined, respectively.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.466-471
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• 1992

To determine the site at which -1 ribosomal frameshifting occurs within the gag-pro overlap of HTL V-I. DNA fragment corresponding to a portion of the gene overlap was cloned into a SP6 vector. The resultant plasmid harbors the hybrid gene consisting of a synthetic gene encoding 5 amino acids derived from chick prelysozyme including the initiator methionine plus 141 nucleotides of gag-pro overlapping region followed by Staphylococcus aurcus protein A gene fragment. In vitro transcription by SP6 RNA polymerase with this DNA template made an abundant amount of single species mRNA. Cell-free translation programmed with the RNA transcribed in vitro yielded a polypeptide of 21 kDal in size. which could be purified into homogeneity by IgG-Sepharose affinity chromatography. In vitro system described in this study must be useful for rapid purification and sequencing of the Gag-Pro transframe protein. allowing to determine the exact frameshift site on mRNA and to identify the tRNA involved in frameshifting event for the expression of pro gene.