• 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.202-205
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• 2000

A gene coding $endo-{\beta}$,-1, 4 glucanase from Actinomyces sp. KNG40 and phytase from Hansenula polymorpha were cloned into Esherichia coli JM101 by using E. coli/Lactobacillus shuttle vector pNZ3004 and pNZ123. The plasmid p3PS(1-4) and p123(1-4) have slpA promoter and slpA signal sequence. So, I constructed expression vectors, p3PS(1-4)Endo, phy and p123(1-4)Endo, phy. These constructed vector was transformed in target host Lactobacillus gasseri and reutri. These transformed host expressed endoglucanase and phytase as extracellular fraction. In the enzyme activity of the same vector, host L, gasseri was higher activity than L. reuteri. This indicates that L. gasseri recongnize promoter and signal sequence very well.

• 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.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.953-962
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• 2015

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, costeffectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.17-20
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• 1999

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.

• 한국미생물·생명공학회지
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• 제35권3호
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• pp.184-190
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• 2007

대략 2 kb의 크기를 가진 Pichia pastoris phosphoglycerate kinase gene (PGK1)의 프로모터부분을 266bp의 작은 크기로 최소화하여 P. pastoris에 있어 episomal의 새로운 항시적 발현벡터를 제조하였다. P. pastoris의 새로운 항시적 발현벡터를 개발하기 위하여 기존의 Pichia발현벡터인 pGABZB의 GAP프로모터부분을 연속적으로 일정 부분이 절단된 PGK1프로모터에 beta-galactosidase유전자가 결합된 부분으로 치환하였다. LacZ유전자를 reporter유전자로 사용하였을 때에 PGK1프로모터의 발현세기는 다른 항시적 프로모터인 GAP프로모터 보다는 낮았지만 TEF1프로모터 보다는 높았다. 본 논문에서 PGK1 프로모터의 불필요한 부분을 제거함으로서 Pichia에서 외래발현을 위한 새로운 episomal발현벡터인 pPGKZ-E를 제조하였으며 이 것은 P. pastoris에 있어 발현세기를 선택할 수 있는 발현벡터선택의 폭을 넓게 하였다.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2019년도 춘계학술대회
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• pp.533-536
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• 2019

재조합 배큘로 바이러스는 배양 된 곤충 세포에서 이종 유전자를 발현하는데 널리 사용된다. 재조합 배큘로 바이러스는 광범위한 포유류 세포 유형에서 재조합 단백질의 발현을위한 유전자 전달 벡터로서 작용할 수있다. 바큘로 바이러스 시스템은 안전성, 대규모 및 높은 수준의 유전자 발현 관점에서 중요한 이점을 갖는다. 본 연구에서는 pOPINEneo-3C-GFP 벡터로부터 재구성 된 바큘로 바이러스 벡터를 사이토 메갈로 바이러스 (CMV) 프로모터, 강화 된 녹색 형광 단백질 (EGFP) 및 p53과 NcoI 및 XhoI로 재조합시켰다. 이러한 재조합 벡터를 다양한 세포 및 세포주에 감염시켰다. 이와 같이 개발 된 바큘로 바이러스 벡터는 재조합 유전자의 전이 및 발현을 통상적 인 벡터와 비교하여 분석 하였다. 이러한 결과는 바큘로 바이러스 벡터가 대조군 벡터보다 전이 및 전이에서 더 높은 효율을 갖는다는 것을 시사한다. 본 연구는 과학 기술부, 한국 정보 기술 진흥 기금 (MSIP)이 후원하는 한국 연구 재단(NRF)을 통해 중견 연구원 프로그램 (NRF-2016R1A2B4016552)을 통해 지원되었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제3권1호
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• pp.13-18
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• 2001

The role of polyhedrin in the polyhedra production in baculovirus Autograha californica Nucelopolyhedro-sisvirus (AcNPV) was studied by over-expression of AcNPV polyhedrin or heterologous polyhedrin from Bombyx mori (Bm) NPV or Spodoptera exigua (Se) NPV. The transfer vectors containing additional polyhedrin from AcNPV, BmNPV, or SeNPV were constructed and cotransfected with bacmid bApGOZA into Sf9 cells. The resulting recombinants, designated as vApAcPol, vApBmPol, and vApSePol were tonstructed, and the polyhedra production of the recombinant was characterized. All of the recombinants produced polyhedra in the nucleus, and the polyhedrin was over-expressed. Among three recombinants, vApAcPol and vApBmPol were discriminated by their larger polyhedra size than that of wild type AcNPV, and vApSePol also produced larger polyhedra than wild type SeNPV polyhedra.

• The Plant Pathology Journal
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• 제21권1호
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• pp.13-20
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• 2005

Chlorella viruses are large icosahedral, plaque-forming, dsDNA viruses that infect certain unicellular, chlorellalike green algae. The genomic DNA of over 300 kb contains many useful genes and promoters. Over 40 chlorella viruses have been isolated from fresh water in Korea since 1998. The viruses were amplified initially in chlorella strain NC64A, and pure isolates were obtained by repeated plaque isolation. SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. The tRNA-coding regions of 8 chlorella viruses were cloned and sequenced. These viruses contain 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which has a 1039-bp spacer in a cluster of 11 tRNA genes. Promoter regions of several early genes were isolated and their activities were analyzed in transformed chlorella. Some promoters showed stronger activity than commonly used CaMV 35S promoter and chlorella transformation vectors for heterologous protein are beings constructed using these promoters.

• Journal of Plant Biotechnology
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• 제46권4호
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• pp.323-330
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• 2019

The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.