• 한국자원식물학회지
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• 제30권1호
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• pp.1-7
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• 2017

본 연구는 항암 단백질로 알려진 lunasin peptide의 산업적 활용성을 높이고자 lunasin peptide를 생산할 수 있는 시스템을 개발하고, 생산된 lunasin peptide가 식물유래 lunasin peptide의 생리활성을 가지는지 chromatin binding 활성과 histone acetylation 억제활성을 통해 평가하였다. 그 결과 E. coli와 P. pastoris를활용하여 재조합 lunasin peptide를 생산했으며, 생산 된 재조합 lunasin 펩타이드가 식물유래 lunasin peptide의 chromatin binding 활성과 histone acetylation 억제활성을 나타냄을 확인할 수 있었다. 따라서, 본 실험 연구의결과를 토대로 lunasin 펩타이드의 대량생산이 진행된다면 천연물 유래 생리활성 물질로서 효과적이면서도 안전한 기능성 식품소재로의 산업적 활용이 가능할 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.587-601
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• 2013

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

• KSBB Journal
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• 제13권3호
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• pp.325-330
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• 1998

The methylotrophic yeast, Pichia pastoris, is known to be a potential host to offer many advantages for production of recombinant proteins. Fermentation parameters were optimized to enhance the heterologous ${\beta}$-galactosidase productivity with P. pastoris. Optimum concentration of methanol, used as inducer, was observed to be 8 g/L and the extent of repression of AOX1 promoter by glycerol was lower than by glucose. The degradation of the gene product ${\beta}$-galactosidase by protease was inhibited as the pH increased from 5 to 8 and the yeast extract(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%). Induction method, in which methanol is just added to fermentation medium without centrifugation, was found to be as much effective as the one with centrifugation.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.171-174
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• 2005

20 g/L peptone, 20 g/L dextrose, 10 g/L yeast extract에 100 mg/L zeocin을 첨가하여 동일하게 전배양 한 재조합 Pichia pastoris X-33/pBPT44를 각기 다른 탄소원이 든 배지에 배양하면서 12시간 간격으로 샘플을 채취하여 배양시간에 따른 세포성장, pH, 각 탄소원에 따른 PLC 생산량 등을 측정하였다.

• KSBB Journal
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• 제16권1호
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• pp.15-23
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• 2001

The development of expression systems for heterologous proteins has been greatly demanded not only for the study of the structure/function relationships of these proteins but also for their biotechnological and pharmaceutical applications. During the past decades, the methylotrophic yeast Hansenula polymorpha and Pichia pastoris have drawn attention as one of promising hosts for the production of a variety of heterologous proteins. The increasing popularity of H. polymorpha and P. pastoris as the host systems can be attributed to the several advantages over the traditional yeast Saccharomyces cerevisiae, such as the availability of very strong and tightly regulated promoters from the enzymes involved in the metabolism of methanol, a very high-cell density even on simple mineral media, and a high stability of expression plasmids. Furthermore, it has been observed that glycoproteins from these two yeasts are less hyperglycoylated compared to those from S. cerevisiae. Despite substantial similarities as methylotrophic yeasts, however, these two expression systems have some unique features distinguished from each other. In this paper we present a brief overview on the present status of the expression systems developed in methylotrophic yeast, mainly focusing on the similarities and differences between the H. polymorpha and P. pastoris systems.

• 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.

• 한국미생물·생명공학회지
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• 제38권1호
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• pp.40-45
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• 2010

Agarose의 $\beta$-1,4결함을 분해하는 Zobellia galactanivorans 유래의 $\beta$-agarase 유전자(agaB)는 클로닝 되었고, AOX1(alcohol oxidase 1, methanol inducible) promoter 하류에 Saccharomyces cerevisiae mating factor alpha-1 secretion signal($MF{\alpha}1$)를 연결하여 $MF{\alpha}1$-AgaB를 구축하였다. 구축된 plasmid pPIC-AgaB(9 kb)를 Pichia pastoris genome에 HIS4 gene 위치에 integration하였고, colony PCR을 통해 확인하였다. Methanol 첨가 배지에서 자란 형질전환체는 iodine solution의 첨가에 의해 red halos를 보였으며, P.pastoris에서 agaB의 효율적 분비 발현을 확인하였다. SDS-PAGE와 zymographic analysis에서 $\beta$-agarase의 분자량은 약 53 kDa으로 추정되었으며, 15% 정도의 N-linked glycosylation이 일어났음을 알 수 있었다. P.pastoris GS115/pPIC-AgaB의 48시간 baffled flask culture에서 세포외 $\beta$-agarase의 활성은 각각 0.1, 0.5, 1% methanol의 유도에 의해 1.34, 1.42 그리고 1.53 units/mL의 활성을 보였다. 대부분의 $\beta$-agarase의 활성은 세포 외에서 관찰되었고, 분비효율은 98%였으며 분비시의 glycosylation에 의해 열안정성도 증가되었다.

• 한국미생물·생명공학회지
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• 제30권4호
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• pp.305-311
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• 2002

Aspergillus ficuum 유래 acetyl xylan esterase(AXEase) 유전자(AXE)를 Pichia pastoris에서 과발현ㆍ분비 생산하기 위해 AOXI promoter와 mating factor $\alpha$-1 분비신호서열 하류에 AXE를 연결한 염색체 삽입 발현계(pPICZ$\alpha$C-AXE, 4.6 kb)를 구축하였다. 이것을 SacI으로 절단한 뒤 P. pastoris의 염색체 DNA 5'AOX1 부위에 삽입시켰다. 형질전환된 P. pastoris 균주를 메탄을 배지에서 플라스크 회분배양한 결과, 배양 36시간 때의 건조균체농도는 6 g-DCW/1, AXEase 총 발현량은 77 unit/ml이었다. 최적화된 methanol과 histidine 공급방법을 채용한 유가배양시 균체농도는 97 g-DCW/1, AXEase 총발현량은 930 unit/m1로 크게 증가하였다. 효소활성의 90% 이상은 배양 상등액에 존재하였으며, 상등액 단백질의 80%이상이 AXEase 단백질(33.5 kDa)였다. 이러한 결과는 9.8 g/l의 AXEase 단백질을 배양 상등액으로 대량 분비ㆍ생산할 수 있음을 의미한다.

• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.461-468
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• 2016

In recent years, various naturally occurring defence peptides such as plectasin have attracted considerable research interest because they could serve as alternatives to antibiotics. However, the production of plectasin from natural microorganisms is still not commercially feasible because of its low expression levels and weak stability. A tandemly arrayed plectasin gene (1,002 bp) from Pseudoplectania nigrella was generated using the isoschizomer construction method, and was inserted into the pPICZαA vector and expressed in Pichia pastoris. The selected P. pastoris strain yielded 143 μg/ml recombinant plectasin (Ple) under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Ple was estimated by SDS-PAGE to be 41 kDa. In vitro studies have shown that Ple efficiently inhibited the growth of several gram-positive bacteria such as Streptococcus suis and Staphylococcus aureus. S. suis is the most sensitive bacterial species to Ple, with a minimum inhibitory concentration (MIC) of 4 μg/ml. Importantly, Ple exhibited resistance to pepsin but it was quite sensitive to trypsin and maintained antimicrobial activity over a wide pH range (pH 2.0 to 10.0). P. pastoris offers an attractive system for the cost-effective production of Ple. The antimicrobial activity of Ple suggested that it could be a potential alternative to antibiotics against S. suis and S. aureus infections.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.467-473
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• 2010

To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.