• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.827-831
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• 2007

Myostatin is a member of the transforming growth factor-${\beta}$(TGF-${\beta}$ super-family. It acts as a negative regulator for skeletal muscle growth. Myostatin mutations are characterized by a visible, generalized increase in muscle mass in double muscled cattle breeds. To understand the biochemistry and physiology of the Chinese Yellow bovine myostatin gene, we report here for the first time expression of the gene in Escherichia coli (E. coli). Primers of the myostatin gene of Chinese Yellow Cattle were designed on the basis of the reported bovine myostatin mRNA sequence (Gen-Bank Accession No. NM005259) and optimized for E. coli codon usage. XhoI and EcoRI restriction enzyme sites were incorporated in the primers, and then cloning vector and expression vector were constructed in a different host bacterium. The expressed protein had a molecule mass of about 16 kDa as determined by SDS-PAGE under reducing conditions. The expressed protein reacted specifically with myostatin monoclonal antibody on immunoblots. Our studies should lead to the investigation of the differences in myostatin genes of various cattle and could benefit human health and food animal agriculture.

• Korean Journal of Microbiology
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• v.30 no.3
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• pp.149-153
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• 1992

Cloning of the gene encoding extracellular .betha.-lactamase from Streptomyces sp. SMF13 in a plasmid pIJ702 and expression of the gene in Streptomyces invidans were carried out. Optimal conditions for the formation of protoplasts of S.lividans and the regeneration of the protoplasts were evaluated. Streptomyces sp. SMF-13 was selected as a donor strain of .betha.-lactamase gene and totla DNA of the strain was partially digested with Sau3A I. DNA fragments ranged from 4kb to 10 kb were ligated to pIJ702 AT Bgl II site and then the ligated DNAs were transformed to the protoplasts of S, livivans. The transformation efficiency was $2 *10^{3}$ .$\mu$g DNA for the ligated DNA mixture. One colony among a thousand colonies regenerated showed extracellular .betha.-lactamase and the size of the inserted DNA fragment was estimated to be 3.94 kb. The .betha.-lactamase activity in the culture broth of the recombinant strain was maximum at 3 days culture to be 1.0 unit/ml.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.2
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• pp.115-122
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• 2007

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.251-255
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• 1991

A 16 kilobase (kb) HindIII fragment of alkalophilic Bacillus sp. YC-335 containing a gene for xylanase synthesis was inserted at the HindIII site of pBR322 and cloned in Escherichia coli HB101. After subcloning of recombinant plasmid pYS52, the 1.5 kb fragment was found to code for xylanase activity, and the hybrid plasmid was named pYS55. The DNA insert of the plasmid was subjected to restriction enzyme mapping, which showed that pYS55 had single site for PuvII and SstI in the 1.5 kb insert fragment. Southern hybridization analysis revealed that the cloned gene was hybridized with chromosomal DNA from alkalophilic Bacillus sp. YC-335. About 64% of the enzyme activity was observed in the extracellular and periplasmic space of E. coli HB10l carrying pYS55.

• 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.21 no.12
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• pp.1294-1298
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• 2011

A metK gene encoding S-adenosyl-L-methionine synthetase was cloned from the non-Streptomyces actinomycetes, Actinoplanes teichomyceticus ATCC31121. In order to evaluate the effect of the metK expression on antibiotic production in actinomycetes, an expression vector harboring the metK gene was constructed and introduced into Streptomyces lividans TK24 and A. teichomyceticus, and the antibiotic production of the exconjugants was assessed. As a result, it was determined that the expression of metK induced 17-fold and 2.2-fold increases in actinorhodin production from S. lividans TK24 and teicoplanin production from A. teichomyceticus, respectively, compared with the control strains.

• Proceedings of the Korean Society of Veterinary Clinics Conference
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• 2008.10a
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• pp.162-162
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• 2008

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• International Journal of Industrial Entomology and Biomaterials
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• v.14 no.2
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• pp.69-74
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• 2007

As the genome of B. mori is available in GenBank and the EST database of B. mori is expanding, identification of novel genes of B. mori was conceivable by datamining techniques and bioinformatics tools. In this study, we used the in silico cloning method to get the 6-Phosphogluconolactonase (6PGL) gene of B. mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and prokaryotic expression. The 6PGL cDNA comtains a 702 bp ORF. The deduced protein has 233 amino acid residues, with the predicted molecular weight of 25946. 72 Da, isoelectric point of 5.41, and contains conserved NagB domains. This gene has been registered in GenBank under the accession number EF198104.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.65-71
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• 2001

A gene from Paenibacillus sp. KCTC 8848P encoding ${\beta}-amylase$ was cloned and expressed in Escherichia coli. The Paenibacillus ${\beta}-amylase$ gene cosisted of a 2,409-bp open reading frame without a translational stop codon, encoding a protein of 803 amino acids. The presumed ribosime-binding site, GGAGG, was located 10 bp upstream from the TTG initiation codon. The deduced amino acid sequence of the ${\beta}-amylase$ gene had a 95% similarity to the ${\beta}-amylase$ of Bacillus firmus. The ${\beta}-amylase$ gene was introduced into wild-type strains of Saccharomyces cerevisiae using a linearized yeast integrating vector containing a geneticin resistance gene and its product was secreted into the culture medium.