• Journal of Microbiology and Biotechnology
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• 제4권1호
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• pp.1-6
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• 1994

Continuous culture was carried out with a recombinant Escherichia coli W3110/pCR185, which encodes trp-operon enzymes when the temperature is shifted from $37^{circ}C\;t;42^{\circ}C$. Under derepressed condition of $42^{\circ}C$. plasmlid stability and gene expression were analysed as function of the dilution rate. The stability of plasmid increased with the dilution rate, but maximal levels of gene expression (tryptophan concentration) and plasmid DNA content were obtained at the lowest dilution rate, $0.075\;hr^{-1}$. The plasmid instability, observed at low dilution rates, could be explained by the unbalanced biosynthetic state of the recombinant cell harboring a high copy number of plasmid.

• BMB Reports
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• 제41권10호
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• pp.739-744
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• 2008

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding $\beta$-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing $\beta$-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.1-14
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• 2004

More than twenty years have passed since the approval of the first recombinant DNA product for therapeutic use (recombinant human insulin, 1982). However, the biotechnology industry is still facing a shortage of manufacturing capacity due to the increasing demand of therapeutic proteins. This demand has prompted the search for a growing number of biological production systems but, nevertheless, the Gram-negative bacterium Escherichia coli remains one of the most attractive production hosts. This review highlights the most important features and developments of plasmid vector design, emphasizing the different reported strategies for improving the expression and secretion of heterologous proteins using the cellular machinery of E. coli.

• 생명과학회지
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• 제18권3호
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• pp.422-425
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• 2008

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated zinc-dependent endoproteinase involved in extracellular matrix remodeling. MT1-MMP hydrolyzes ECM proteins like collagen and is involved in cancer cell migration and metastasis. Caveolins are integral membrane proteins and play a role in formation of caveolae, specialized membrane microdomains involved in clathrin-independent endocytosis. Recombinant MT1-MMP was transiently expressed in PANC-1 cells. Cells expressing recombinant MT1-MMP were able to hydrolyze collagen and migrate on collagen coated trans-well. Both subjacent collagen degradation and the cell migration conferred by recombinant MT1-MMP were inhibited by co-transfection of plasmids containing caveolin-1 cDNA. The results support that MT1-MMP is localized in lipid raft of the membrane and MT1-MMP activities in invasive cells could be inhibited by caveolin.

• KSBB Journal
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• 제20권2호
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• pp.112-115
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• 2005

본 연구는 장시간의 산업적인 연속 배양에서 문제가 되는 플라즈미드 불안정성을 극복하기 위해서 박테리오 파아지람다를 벡터로 이용하였다. 또한, 벡터의 안정성과 생산성을 높이기 위해서 용균과 ${\lambda}DNA$패키징이 결핍된 돌연변이 람다를 선별하였다. 이 돌연변이 람다는 온도 전환에 의해서 단백질이 생산되는 온도 민감성 돌연변이 cI유전자를 가지고 있기 때문에 재조합 단백질 생산, ${\lambda}DNA$ 복제,숙주 세포의 안정성 등이 lytic상태로의 전환을 유도하는 온도에 영향을 받게 된다. $36^{\circ}C$의 배양 온도는 lytic으로 전환이 잘 되지 않았고, $40^{\circ}C$ 이상의 배양 온도는 완전한 lytic상태를 유도하였다. 그러나$42^{\circ}C$의 배양 온도에서는 생산성이 감소되는 온도 저해 효과가 관찰되었다. 온도가 증가할수록 박테리오 파아지가 들어 있지 않는 대장균의 수는 증가하였고,이것은 새로운 파아지를 만들 수 있는 박테리오 파이지를 사용하여 재감염을 시키면 완화될 수 있을 것으로 예상된다. 결과적으로 단위 세포당 발현량은 $40^{\circ}C$에서 최대를 나타내었고, 안정성이나 총 발현량의 관점에서는 $38^{\circ}C$가 최적의 온도로 관찰되었다.

• BMB Reports
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• 제40권4호
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• pp.459-466
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• 2007

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

• BMB Reports
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• 제36권6호
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• Journal of Microbiology and Biotechnology
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• 제1권1호
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• pp.17-21
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• 1991

A gene coding for ${\beta}-xylosidase$ in alkalophilic Bacillus sp. YC-335 isolated from soil was cloned into Escherichia coli HB101 using plasmid pBR322. The recombinant plasmid pYK40 was isolated, and the cloned HindIII fragment was 15 kilobases (kb). To reduce the size of the inserted DNA fragment of pYK40, the 15 kb HindIII fragment was subjected to a series of subclonings. A 6 kb subfragment was found to code for ${\beta}-xylosidase$ activity, and the recombinant plasmid was named pYK44. Southern hybridization analysis revealed that the cloned gene hybridized with 3.5 kb, 1.5 kb, and 1.0 kb of HindIII cleaved chromosomal DNA from Bacillus sp. YC-335. ${\beta}-xylosidase$ activity produced by recombinant E. coli was found to be 11 times higher than that produced by Bacillus sp. YC-335. Xylan was required to induce the production of ${\beta}-xylosidase$ in Bacillus sp. YC-335.

• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.350-355
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• 2010

Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.

• 한국미생물·생명공학회지
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• 제22권6호
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• pp.607-613
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• 1994

The Bacillus stearothermophilus arfI gene encoding a-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate. The $\alpha$-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or $o$-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.