• International Journal of Industrial Entomology and Biomaterials
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• 제30권2호
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• pp.50-57
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• 2015

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has type-specific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti-His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

• Parasites, Hosts and Diseases
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• 제42권4호
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• pp.195-200
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• 2004

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.

• 식물조직배양학회지
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• 제27권4호
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• pp.283-297
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• 2000

Seeds are an ideal repository for recombinant proteins in molecular farming applications. However, in order to use plant seeds efficiently for the production of such proteins, it is necessary to understand a number of fundamental biological properties of seeds. This includes a full understanding of promoters which function in a seed-specific manner, the subcellular targeting of the desired polypeptide and the final form in which a protein is stored. Once a biologically active protein has been deposited in a seed, it is also critical that the protein can be extracted and purified efficiently. In this review, these issues are examined critically to provide a number of approaches which may be adopted for production of recombinant proteins in plants. Particular attention is paid to the relationship between subcellular localization and protein extraction and purification. The robustness and flexibility of seed-based production is illustrated by examples close to or already in commercial production.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.750-753
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• 2001

Proteomics is a formalized approach for obtaining a rapid snap-shot of the protein complement of a tissue, cell or cell component. Such an approach is powerful in that it allows a parallel assessment of temporal protein fluxes. This is an important concept in view of the dynamic nature of protein expression. Undoubtedly, changes in protein expression are essential in any study aimed at investigating cellular networks. In this study, we analyzed and compared the proteomes of recombinant E. coli strain before and after induction. Proteome expression patterns of recombinant E. coli were resolved on 2D-gels, and the variations in the relative expression level of particular proteins were examined using software-aided protein quantification tool. We observed above 800 spots on a 2D-gel using Melanie II software. Many proteins which involved in chaperones were significantly up-regulated in recombinant E. coli. Therefore, it could be concluded that the expression of recombinant protein in E. coli acted as a stress to the cells, which change cells ability to synthesize proteins and induced the expression of various protective proteins.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• KSBB Journal
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• 제24권1호
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• pp.96-100
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• 2009

본 연구에서는 저온에서의 재조합 단백질 생산성 향상을 위하여 저온에서 RNA 샤페론 활성을 지닌다고 알려진 CspA 단백질의 발현이 서로 다른 온도에서 대장균의 성장 및 GFP의 발현 속도에 어떻게 영향을 미치는지를 살펴보았다. $20^{\circ}C$, $25^{\circ}C$, $37^{\circ}C$에서는 세포 성장 및 GFP의 생산이 CspA의 발현에 영향을 받지 않았으나, $15^{\circ}C$에서는 GFP의 총 생산성이 CspA의 동시 발현에 의해 향상되었으며 이는 세포 성장 속도의 향상에 기인함을 확인하였다. 결론적으로 CspA의 발현은 $15^{\circ}C$에서 세포 당 재조합 단백질 생산량의 증가에는 영향을 미치지 않으나, 즉 재조합 단백질의 번역 효율에는 큰 영향을 미치지 않으나, 대장균 성장 속도에 영향을 미치며, 이를 통해 재조합 단백질의 총 생산량 향상을 유도 할 수 있을 것으로 기대된다.

• 대한수의학회지
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• 제37권4호
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• pp.875-882
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• 1997

To produce the recombinant fibronectin-binding protein(FnBP) for development of subunit vaccine against Staphylococcus aureus. The fnbp gene was amplified from the chromosomal DNA of S aureus KNU 196 strain using the polymerase chain reaction, and cloned into pGEX-4T-2. Then, the recombinant FnBP fused with glutathione-S-transferase was produced in E coli, purified by affinity chromatography, and identified its antigenicity and immunogenicity by Western blot. The recombinant FnBP produced in this study is considered to have the same property of native FnBP purified from S aureus, and is expected to be useful as a candidate for S aureus subunit vaccine.

• 한국미생물·생명공학회지
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• 제21권5호
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• pp.504-510
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• 1993

The effect of AcNPV infection conditions such as serum concentration, pH, CaCl2, lysosomotropic agent, cell density at infection, agitation, aeration and nutritional supplementattion on recombinant protein production in Spodoptera frugiperda 21 cells was investigated using tissue culture flask, bottle and spinner flask. It was shown that serum, CaCl2, pH and cell density at infection affected recombinant production. The lysosomotropic agent did not significantly influence recombinant protein production.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• 미생물과산업
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• 제19권4호
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• pp.14-26
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• 1993

Optimizing protein production in recombinant E. coli strains involves manipulation of genetic and environmental factors. In designing a production system, attention must be paid to gene expression efficiency, culture conditions and bioreactor configuration. Although not much emphasis was given to the physiology of host strains in this review, an understanding of the relationship between the physiology of host cell growth and the overproduction of a cloned gene protein is of primary importance to the improvement of the recombinant fermentation processes. Sometimes it is desirable to make use of gene fusion systems, e.g. protein A, polypeptide, gutathione-S-transferase, or pneumococcal murein hydrolase fusion, to facilitate protein purification.