• Journal of Microbiology and Biotechnology
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• v.15 no.2
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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.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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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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• v.16 no.7
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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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• v.25 no.3
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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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• v.36 no.4
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• pp.377-382
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• 1993

Rice (Oryza saliva L.) calli containing both embryogenic callus (EC) and non embryogenic callus (NEC) regions were initiated from the mature seed on MS medium supplemented with 2.0 mg/L 2,4-D, 0.5 mg/L kinetin. The calli were developed into two callus type which can be distinguished by visual examination depending on color and appearance. In order to illucidate the polypeptide composition between EC and NEC, the total protein extracted from two types of callus was analysed by electrophoresis. By one-dimesional anlaysis of SDS-PAGE and Isoelectric focusing, several protein bands showed quantitative and qualitative difference in each type of callus. The further analysis of the total protein with two-dimensional electrophoresis showed at least 20 EC specific protein and 10 NE specific protein. Also 3 specific protein spots showing micro heterogeneity of 90, 65, 50 kD were detected in EC, while a series of acidic heterologous protein spots were visualized in NEC.in NEC.

• Journal of Pharmaceutical Investigation
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• v.35 no.2
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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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• v.12 no.1
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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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• v.8 no.2
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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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• v.16 no.1
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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.

• Journal of Applied Biological Chemistry
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• v.53 no.2
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• pp.105-107
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• 2010

To overcome the limitation of E. coli expression system such as inclusion body formation and disulfide bond failure, we tried to express the heterologous protein as a secreted form. We adopted agarase signal peptide (ASP; 23 amino acid residues) from Agarivorans albus YKW-34 which is one of marine bacteia. When we used ASP to express $\beta$-agarase, about 42% activity was detected in media.