• Journal of Microbiology and Biotechnology
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• 제29권6호
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• pp.944-951
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• 2019

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of $80^{\circ}C$ at pH 11.0. The optimum substrate for enzyme activity was $C_{10}$, which is highly stable in methanol solvent. The enzyme was strongly activated by $Ca^{2+}$, $Mg^{2+}$, and strongly inhibited by $Fe^{2+}$ and $Zn^{2+}$. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.531-531
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• 2003

• 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.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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• pp.44-44
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• 2003

Protein disulfide isomerase (PDI) is not only an isomerase catalyzing the formation of native disulfide bond(s) of nascent peptide, but also a molecular chaperone assisting chain folding. We have already reported the structure of a cDNA (bPDl) encoding PDI from Bombyx mori and the function of PDI as foldase in assisting protein folding. (omitted)

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2002년도 Join Meetings of Korean Society of Sericultural Science and Japanse Society of Sericultural Science
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• pp.55-55
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• 2002

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.542-549
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• 2010

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin $\beta$-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at $90^{\circ}C$ for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}$ and $50^{\circ}$, respectively. Specifically, the activity of malate dehydrogenase (MDH) at $85^{\circ}$ was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of pro-carboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

• Journal of Microbiology
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• 제45권5호
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• pp.409-417
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• 2007

Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and $60^{\circ}C$. A broad range of lipase substrates, from $C_4\;to\;C_{18}$ p-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was p-nitrophenyl caproate ($C_6$). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family 1.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, $Ser^{131},\;His^{330},\;and\;Asp^{308}$, which composed the catalytic triad of the enzyme.

• 한국잠사곤충학회지
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• 제44권2호
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• pp.69-73
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• 2002

본 연구는 누에신 단백질의 대량발현을 통해 농업용 소재로서 이용하고자 하는 연구의 일환으로 누에 핵다각체병 바이러스 유래의 pBm10po1-Xa 벡터에 누에신 유전자를 도입하여 누에 배양세포(Bm5) 및 누에 생체에서 발현한 결과 누에신 전사체는 바이러스 접종 후 1일째부터 발현되기 시작하여 5일째에 최대로 발현되었음을 확인할 수 있었고, 누에신 단백질은 3일째부터 발현량이 증가하여 5일째까지 계속 지속적으로 발현되었으나 그 발현량은 전사체에서 처럼 많지 않음을 확인할 수 있었으며, 누에신 단백질의 발현량은 누에 세포에 비해 누에 생체에서 기대만큼 그 발현량이 많지 않았다. 또한 누에신의 베큘로바이러스 발현계 (baculovirus expression vector system, BEVS)를 이용하여 세포내 및 번역후 변형과정을 통하여 세포외로 분비된 누에신의 발현양상을 확인한 결과 세포내에 비해 세포외로 분비시 그 발현량이 현저히 줄어들었음을 확인할 수 있었다. 따라서 베큘로바이러스 발현계를 이용하여 외래 단백질을 생산할 경우 정확한 메카니즘은 밝혀지지 않았으나 강력한 프로모터에 의해 세포내 단백질 생산량은 많은데 비해 정확한 단백질 고차구조 형성을 도와주는 foldase 및 chaperon의 양은 한정되어져 있으므로 정확한 고차구조를 형성하여 세포외로 분비되는 생물학적 활성을 띠는 단백질은 매우 적은 양간이 발현됨을 확인할 수 있었다. 그러므로 추후 누에 신 단백질 대량생산을 위해서는 분비 프로세싱의 해명 및 번역 후 변형과정의 개선을 통한 발현계의 개량이 시급히 요구된다.

• KSBB Journal
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• 제23권3호
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• pp.205-212
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• 2008

본 연구의 목적은 위치특이적 펩타이드 및 단백질을 사용하여 재조합 대장균에서 활성형 인간 상피세포성장인자(hEGF)를 고효율로 발현할 수 있는 방법을 찾아내는 데 있다. 재조합 대장균내 cytoplasm 및 periplasm 영역에서 hEGF의 발현을 위해 각각 세개의 응합 펩타이드 및 단백질을 선정하여 상호 비교하였다. 재조합 대장균에서 hEGF의 발현유도시 대부분 불용성 단백질로 생산되는 현상을 극복하기 위해 cytoplasm영역에서는 ATS, thioredoxin, 리파제를 융합파트너로 사용하였으며 periplasm 영역에서는 foldase인 DsbA와 DsbC, 용융성 고발현 단백질인 maltose binding protein을 선택하여 사용하였다. Periplasm영역에서 발현유도를 시키는 융합단백질의 경우 cytoplasm영역에서의 발현양도 용융성 형태로 고발현 되는 것을 알 수 있었으며 전체적으로 약 2배가량의 용융성 형태로 발현되었다. hECF의 발현율을 가장 높일 수 있는 융합단백 질은 maltose binding protein이었으나 발현된 융합단백질의 24%가 불용성 단백질로 형성되어 활성형 형태로 얻는 데 한계가 있었으며, 활성형 형태로 hEGF의 발현을 위해서는 DsbA를 응합단백질로 사용한 경우에 18.1 mg/L로 가장 높은 발현농도를 보였다. Cytoplasm 영역에서 발현유도를 한 경우에는 ATS와 thioredoxin을 응합파트너로 hEGF를 발현한 경우 용융성 형태로 높은 발현율을 보였다. 특히 ATS와 같은 펩타이드를 N-말단에 융합시킨 경우 불용성을 방지하는 효과를 보여 이황화결합의 불완전성이나 소수성으로 인해 불용성 단백질로 발현되는 기존의 단백질을 활성형 형태로 발현하는데 될 수 있음을 확인할 수 있었다.