• Applied Biological Chemistry
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• 제38권1호
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• pp.7-12
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• 1995

본 연구는 lacZ' 유전자의 promoter 개발을 위하여 착수하였다. lacZ' 유전자의 heterologous promoter I과 II를 효모 염색체의 Bam HI DNA 단편에서 분리하였다. Promoter I의 크기는 2.5 Kb 정도이고 ${\beta}-galactosidase$ 활성은 124.6 U/mg protein이었으며 promoter II의 크기와 효소활성은 4.0 Kb와 168.8 U/mg이었다. 형질 전환체에서의 YEp plasmid 안정성은 52.7%에서 67.4% 정도였다. YEp plasmid로부터 YIp plasmid를 재조합하였으며 이 YIp plasmid는 대장균에서나 효모에서도 발현되었다. 효모로부터 분리한 promoter I과 II는 재조합된 YEp와 YIp plasmid의 promoter로서 이용 가능하였다.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.330-336
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• 2005

Lactococcus lactis A164 is a nisin Z-producing strain isolated from kimchi. Its antimicrobial spectrum has been found to be active against most Gram-positive bacteria tested, yet inactive against Gram-negative bacteria [3]. Accordingly, to overcome this drawback, the current study attempted to express human $\beta$-defensin-l (hBD-l), which kills both Gram-positive and Gram-negative bacteria in L. lactis AI64. When the hBD-l cDNA was introduced using a nisin Z-controlled expression cassette, the L. lactis A164 transformants grew very poorly, due to the bactericidal effect of the expressed hBD-l against the transformants. Therefore, a gene fusion system was designed to reduce the toxicity of the expressed heterologous protein against the host cells. As such, the hBD-l gene was fused to the DsbC- Tag of pET -40b(+), then introduced to L. lactis A 164. The transformants expressed an intracellular 35.6-kDa DsbC-hBD-l fusion protein that exhibited slight activity against the host cells, yet not enough to strongly inhibit the cell growth. To obtain the recombinant hBD-l, the DsbC-hBD-l fusion protein was purified by nickel-affinity column chromatography, and the DsbC-Tag removed by cleaving with enterokinase. The cleaved mature hBD-l exhibited strong bactericidal activity against E. coli JM109, indicating that the recombinant L. lactis A 164 produced a biologically active hBD-I. In addition, the recombinant L. lactis A 164 was also found to produce the same level of nisin Z as the wild-type.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2000년도 추계학술발표대회
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• pp.78-82
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• 2000

The AIDS epidemic continues unabated in many part of the world. After near two decades, no vaccine is available to combat the spread of this deadly disease. Much of the HIV -1 vaccine effort during the past decade has focused on the viral envelope glycoprotein, largely because it is the only protein that can elicit neutralizing antibodies (Nabs). Eliciting broadly cross-reactive Nabs has been a primary goal. The intrinsic genetic diversity of the viral envelope, however, has been one of the major impediments in vaccine development. We have recently completed a comprehensive study examining whether it is possible to elicit broadly acting Nabs by immunizing monkeys with mixtures of envelope proteins from multiple HIV -1 isolates. We compared the humoral immune responses elicited by vaccination with either single or multiple envelope proteins and evaluated the importance of humoral and non-humoral immune response in protection against a challenge virus with a homologous or heterologous envelope protein. Our results show that (1) Nab is the correlate of sterilizing immunity, (2) Nabs against primary HIV -1 isolates can be elicited by the live vector-prime/protein boost approach, and (3) polyvalent envelope vaccines elicit broader Nab response than monovalent vaccines. Nonetheless, our findings clearly indicate that the increased breadth of Nab response is by and large limited to strains included in the vaccine mixture and does not extend to heterologous non-vaccine strains. Our study strongly demonstrates how difficult it may be to elicit broadly reactive Nabs using envelope proteins and sadly predicts a similar fate for many of the vaccine candidates currently being evaluated in clinical trials. We have started to evaluate other vaccine candidates (e.g. genetically modified envelope proteins) that might elicit broadly reactive Nabs. We are also exploring other vaccine strategies to elicit potent cytotoxic T lymphocyte responses. Preliminary results from some of these experiments will be 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.

• The Plant Pathology Journal
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• 제25권3호
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• pp.225-230
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• 2009

Lily mottle virus (LMoV) causes flower quality reduction in Lilium spp. The coat protein gene was RT-PCR-amplified from total RNA extracted from infected lily leaves and the amplified fragment was cloned into the pRSET expression vector tagged with a His-MBP. The plasmid of recombinant coat protein was used to transform an Escherichia coli strain pLysS and was expressed. The coat protein was purified by affinity chromatography using a Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE. The in vitro-expressed protein was used for immunization of mice. The polyclonal and monoclonal antibodies reacted specifically for the detection of LMoV in lily extracts in Western blot. Moreover the monoclonal antibodies reacted with lily extracts in DAS-ELISA with no unspecific or heterologous reactions against other non-serologically related viruses, but the polyclonal antibodies revealed a weak reaction against both infected lily and healthy control.

• Journal of Plant Biology
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• 제36권4호
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• pp.377-382
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• 1993

벼(Oryza sativa L.) 캘러스는 2.0 mg/L 2,4-D와 0.5 mg/L kinetin이 첨가된 MS 배지에서 성숙종자로부터 유도되었으며 embryogenic callus(EC)와 nonembryogenic callus(NEC)는 색깔과 외부형태에 의해 경시적으로 선별되었다. EC와 NEC의 전체 단백질로부터 SDS-PAGE와 등전점 전기영동에 의한 전기영동적 분석은 EC와 NEC의 각각에 대해 특이적, 양적인 차이를 보여주었다. 또한 EC와 NEC의 2차원 전기영동 분석은 약 20여개 이상의 EC 특이단백질과 10여개의 NEC 특이 단백질 양상을 보여주었으며, 아울러 EC 특이적인 90, 65, 50 kD의 단백질은 microheterogeneity를 보여주는 반면, NEC에서는 분자량의 변이가 큰 일련의 산성 이질단백질군을 보여주었다.

• Journal of Pharmaceutical Investigation
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• 제35권2호
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• pp.75-80
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• 2005

A promising application of Lactococcus lactis is its use as live vehicles for production and delivery of heterologous proteins of vaccines and therapeutic substances. Because L. lactis has GRAS ('generally regarded as safe') status, we tested whether L. lactis could function as the carrier of the Ll protein of human papillomavirus (HPV) type 16. The RNA level expression of Ll gene was detected in L. Lactis. The Ll protein was expressed in L. lactis with Ll gene. The growth of strains L. lactis with an empty plasmid (pAMJ328) and L. lactis with Ll-encoding plasmid (pAMJ328-Ll) was slightly decreased in comparison with the growth of strains L. lactis (wild type). However, all the three strains of L. lactis maintained the ability to ferment sugars primarily into lactic acid, indicating that Ll protein did not affect the biochemical property of L. lactis. These results suggest that L. lactis, capable of carrying Ll protein, might be further developed as a biocompatible oral protein delivery system.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.93-103
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• 1999

Recent advances in the biotechnology of ruminal bacteria have been made in the characterization of enzymes involved in plant cell wall digestion, the exploration of mechanisms of gene transfer in ruminal bacteria, and the development of vectors. These studies have culminated in the introduction and expression of heterologous glucanase and xylanase genes and a fluoroacetate dehalogenase gene in ruminal bacteria. These recent studies show the strategy of gene and vector construction necessary for the production of genetically engineered bacteria for introduction into ruminants. Molecular research on proteolytic turnover of protein in the rumen is in its infancy, but a novel protein high in essential amino acids designed for intracellular expression in ruminal organisms provides an interesting approach for improving the amino acid profile of ruminal organisms.

• The Korean Journal of Physiology and Pharmacology
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• 제8권2호
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• pp.111-115
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• 2004

Roundabout (Robo) is the transmembrane receptor for slit, the neuronal guidance molecule. In this study, the tyrosine phosphorylation of Robo was observed in Robo-transfected human embryonic kidney cells and developing rat brains, and found to be increased by the treatment with protein kinase A activator, forskolin. In contrast, protein kinase C activation by phorbol-12-myristate-13-acetate decreased the phosphorylation of Robo. Intracellular calcium was required for the tyrosine phosphorylation. Furthermore, the transfection of an Eph receptor tyrosine kinase dramatically enhanced the tyrosine phosphorylation. These findings indicate that the tyrosine phosphorylation of Robo is regulated by multiple mechanisms, and that Eph receptor kinases may play a role in the regulation of tyrosine phosphorylation of Robo in the rat brain.

• KSBB Journal
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• 제16권1호
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• pp.1-10
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• 2001

Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.