• Korean Journal of Plant Resources
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• v.30 no.1
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• pp.1-7
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• 2017

In this study, we produced the recombinant lunasin peptide using E. coli and P. pastoris, and evaluated biological activity of the recombinant lunasin peptide. Lunasin peptide was produced from E. coli transfected with pPGEX-lunasin expression vector and P. pastoris GS115 transfected with pPIC-lunasin expression vector. These recombinant lunasin peptides were similar to the synthetic lunasin peptide in the identification by LC-ESI-MS. In addition, the recombinant lunasin peptide from E. coli and P. pastoris was bound in the chromatin, and inhibited histone acetylation and the activity of histone acetyltransferase. These findings suggest that the production of the lunasin peptide using E. coli and P. pastoris will be useful for industrial utilization of lunasin peptide.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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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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• v.13 no.3
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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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• 2005.04a
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• pp.171-174
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• 2005

The Pichia pastoris expression system is widely used for e production of recombinant proteins. In this study, we investigated the characteristics of cell growth and PLC production of recombinant Pichia pastoris. Especially, severed carbon sources like glycerol, glucose, sucrose, mannose, dextrose, xylitol, lactic acid, and acetic acid etc. were used to fermentations. The PLC activity was measured photometically.

• KSBB Journal
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• v.16 no.1
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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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• v.22 no.9
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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.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.40-45
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• 2010

The gene encoding $\beta$-agarase (agaB) which hydrolyzes $\beta$-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal ($MF{\alpha}1$), in which the transcription of $MF{\alpha}1$-AgaB was under the control of AOX1 (alcohol oxidase 1, methanol inducible) promoter. The constructed plasmid pPIC-AgaB (9 kb) was integrated into HIS4 gene locus of Pichia pastoris genome. Successful integration was confirmed by performing colony PCR. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the active expression of agaB in P.pastoris. By SDS-PAGE and zymographic analysis, the molecular weight of $\beta$-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular $\beta$-agarase reached 1.34, 1.42 and 1.53 units/mL by inducing 0.1, 0.5, and 1% methanol, respectively, at baffled flask culture of P.pastoris GS115/pPIC-AgaB for 48 hr. Most of the enzyme activity was found in the extacellular fraction and the secretion efficiency showed 98%. Thermostability of recombinant $\beta$-agarase was also increased by glycosylation.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.305-311
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• 2002

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ$\alpha$C-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOXI promoter and connected downstream of mating factor u-1 signal sequence. The plasmid linearized by Sacl was integrated into the 5'AOXI region of the chromosomal DNA of P. pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/1 and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/1 of AXEase protein was produced in the extracellular medium.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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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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• v.20 no.3
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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.