• 대한수의학회지
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• 제43권3호
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• pp.449-456
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• 2003

The VP2 gene of aquatic birnavirus, Korean isolate (GC-1) was cloned and expressed using the baculovirus expression system. The VP2 gene and VP2 partial gene, which contained a neutralizing epitope, were constructed for recombinant transfer vectors, for baculovirus expression. The expressed recombinant proteins were confirmed by indirect immuno fluorescence antibody (IFA), SDS-PAGE and Western blot. The level of expression was checked at regular time using IFA and Western blot. To measure the neutralizing activity of recombinant proteins against GC-1 strain, the antisera against recombinant proteins were produced by using guinea pigs. The result showed that the antisera neutralized the GC-1 strain. However, the neutralizing titer was higher in antisera against the VP2 gene expressed recombinant protein than that of VP2 partial gene recombinant protein.

• Journal of Microbiology
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• 제35권4호
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• pp.341-346
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• 1997

In order to express recombinant porcine TGF-${\beta}1$ protein in a baculovirus expression system the entire TGF-${\beta}1$gene containing extra amino acids at the N terminus was cloned into pFBa and pFBb of the Bac-To-$Bac^{TM}$ baculovirus expression system. One of the clones contained 106 extra amino acids and was designated pFBa-106 TGF-${\beta}1$, and the other had 28 extra amino acids and was designated pFBb-28 TGF-${\beta}1$. The orientation of the gene was identified with restriction enzyme mapping and PCR with internal TGF-${\beta}1$ primers. Sf-9 cells were infected at a m.o.i. of 10 by the recombinant viruses generated from the two expected sizes of 55 kD and 46.4kD. these precursor forms of TGF-${\beta}1$ with a polyclonal antibody against human TGF-${\beta}1$. No mature form of TGF-${\beta}1$ protein was detected on SDS gels and an immunoblot indicated that TGF-${\beta}1$ precursor is not properlu processed in insect cells.

• KSBB Journal
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• 제10권3호
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• pp.317-322
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• 1995

유전자 재조합 단백질 생산을 위한 곤충세포- 배쿨로바이러스 시스템에 있어서, 바이러스에 감염시 키는 시기가 늦을수록 재조합 단백질의 발현이 낮게 나타났다. 이것은 높은 세포농도일수록 단위세포당 낮은 발현율을 의미하므로 재조합 단백질 생산을 위 해 감염시기에 대한 최적화의 필요성을 보여주며, 재조합 단백질의 최대 생산생을 위한 최적 감염시기 의 존재를 실험적으로 업증하였다. 또한 배지에 5% 누에 체액을 보강함으로써 발현율이 증가하였고, 이 것은 누에 체액 첨가로 인해 세포내 바이러스 숫자가 증가하고 감염 후 세포의 생존성이 오래 유지되 는 것에 기 인하는 것으로 생각된다. 고농도 세포배 양을 위해 yeastolate를 첨가하고, 재조합 단백질 발 헌을 위해 누에 체액을 첨가함으로써 ${\beta}$-galactosidase의 생산이 10배 정도 증가하였다.

• 약학회지
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• 제46권4호
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• pp.270-275
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• 2002

The lectin gene from rice was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pET26b and expressed it as a fusion protein with polyhistidine sequences in Escherichia coli. The recombinant protein was produced by induction with 0.4 mM isopropyl-$\beta$-D-thiogalactopyranoside at 37$^{\circ}C$ and purified by an immobilized metal affinity chromatography. The recombinant protein was found to have lectin activity by the hemagglutination inhibition assay. The hemagglutination activity of the recombinant protein was optimal at pH 4.0-7.0 and was dependent on $Ca^{2+}$ and Mn$^{2+}$.2+/.

• Molecules and Cells
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• 제25권3호
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• pp.446-451
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• 2008

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.

• Bulletin of the Korean Chemical Society
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• 제33권12호
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• pp.4041-4046
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• 2012

The recombinant expression of proteins has been the method of choice to meet the demands from proteomics and structural genomics studies. Despite its successful production of many heterologous proteins, Escherichia coli failed to produce many other proteins in their native forms. This may be related to the fact that the stresses resulting from the overproduction interfere with cellular processes. To better understand the physiological change during the overproduction phase, we profiled the metabolites along the time course of the recombinant protein expression. We identified 32 metabolites collected from different time points in the protein production phase. The stress induced by protein production can be characterized by (A) the increased usage of aspartic acid, choline, glycerol, and N-acetyllysine; and (B) the accumulation of adenosine, alanine, oxidized glutathione, glycine, N-acetylputrescine, and uracil. We envision that this work can be used to create a strategy for the production of usable proteins in large quantities.

• Bulletin of the Korean Chemical Society
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• 제29권12호
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• pp.2449-2452
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• 2008

• Journal of Plant Biotechnology
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• 제4권3호
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• pp.95-101
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• 2002

Molecular faming has the potential to provide large amounts of recombinant protein for use in diagnostics and as therapeutics. Various strategies have been developed to enhance the expression level, stability, and native folding of recombinant proteins produced in plants. Few investigations into the subcellular distribution of recombinant proteins within plant cells have been published despite the potential to increase the expression level and impact the purification process. This review article discusses the current strategies used for targeting recombinant proteins to various subcellular locations and the advantages of targeting to seed oil bodies for molecular farming applications. Specifically, the affinity capture of antibodies using recombinant oilbodies is discussed.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1026-1031
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• 2006

The optimization of the production of a fusion protein containing the antigenic domain 1 (AD-1) is of a great importance, considering its use in diagnostic tests. The fusion protein is produced by the fermentation of a recombinant strain of Escherichia coli containing the plasmid Mbg58, which expresses the AD-1 (aa 484-650) of human cytomegalovirus glycoprotein B as a fusion protein together with aa 1-375 of ${\beta}-galactosidase$. An important characteristic of promoters (lac and derivatives) used in recombinant protein production in E. coli is their inducibility. Induction by IPTG is widely used for basic research; however, its use in large-scale production is undesirable because of its high cost and toxicity. In this work, studies using different inducers and carbon sources for the production of a fusion protein containing the AD-l were performed. The results showed that lactose could be used as an inducer in the fermentation process for the production of this protein, and that expression levels could exceed those achieved with IPTG. The use of lactose for protein expression in E. coli should be extremely useful for the inexpensive, large-scale production of heterologous proteins in E. coli. Addition of sucrose to the fermentation medium improved the yield of recombinant protein, whereas addition of fructose or trehalose decreased the yield.

• Journal of Microbiology
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• 제37권4호
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.