• 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.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.966-971
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• 2009

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the $Mut^s $ Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.275-287
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• 2000

Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, app58lication of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1185-1191
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• 2006

The expression of a pullulanase gene in Pichia pastoris was investigated. The gene encoding pullulanase was cloned by PCR using the chromosomal DNA of Bacillus naganoensis as the template. The expression vector pPIC9K-Pu was constructed by inserting the pullulanase gene into plasmid pPIC9K and then transformed into Pichia pastoris SMD 1168 by electroporation. Activity determination, SDS-PAGE, and PCR amplification indicated that the gene of the pullulanase from B. naganoensis had successfully been expressed in SMD 1168 and the molecular size of the expressed recombinant product was about 119.9 kDa. This is the first report on the successful expression of the pullulanase from B. naganoensis in P. pastoris. The transformant secreted recombinant pullulanase with the activity of 350.8 IU/ml in shake-flask culture. The properties of the recombinant pullulanase were characterized.

• 한국식품영양과학회지
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• 제26권4호
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• pp.669-674
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• 1997

면역활성, 항균성 등의 기능성을 보여 식품첨가물로 전량 수입에 의존하여 사용되는 human lactoferrin을 진핵세포에서의 생산을 시도하였다. 우선, 항균성을 보이는 lactoferrin에 대하여 생육저해가 없는 host cell에 lactoferrin 유전자를 발현시키고자 lactoferrin에 대한 항균력을 실험한 결과 Pichia pastoris는 생육저해를 일으키지 않아 이를 lactoferrin 생산균주로 선정하였다. Pichia를 숙주로 하는 pHIL-SI expression vector에 lactoferrin 유전자를 삽입 하였을 때 genomic DNA에 유전자가 integration 되었다. 즉, transformant JY-1, JY-2는 PCR(polymerase chain reaction)과 southern blotting에 의하여 2.4Kb의 크기의 HLF(human lactoferrin) 유전자가 삽입되었음을 확인하였다. 유전자 발현을 검토한 결과 transformant JY-1는 immunoblotting에 의하여 lactoferrin 단백질 생산을 확인하였다. 배양시간에 따른 HLF의 생산성을 알아본 결과 48시간 이후에 75KDa의 HLF단백질이 분비됨을 확인하였다

• Asian-Australasian Journal of Animal Sciences
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• 제27권4호
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• pp.574-579
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• 2014

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.269-273
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• 2005

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1202-1207
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• 2012

A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.202-205
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• 2005

Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1363-1370
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• 2016

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector $pPICZ{\alpha}A$, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.