[논문]Pichia PGK1프로모터의 분석과 P. pastoris에 있어 외래단백질발현을 위한 Episomal벡터의 제조

이성재; 홍인표; 백선열; 최신건

Pichia PGK1프로모터의 분석과 P. pastoris에 있어 외래단백질발현을 위한 Episomal벡터의 제조
Deletion Analysis of Pichia PGK1 Promoter and Construction of an Episomal Vector for Heterologous Protein Expression in P. pastoris 원문보기

한국미생물·생명공학회지 = Korean journal of microbiology and biotechnology, v.35 no.3, 2007년, pp.184 - 190

이성재 (강원대학교 생물공학과) , 홍인표 (강원대학교 생물공학과) , 백선열 (강원대학교 생물공학과) , 최신건 (강원대학교 생물공학과)

초록
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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.

Abstract ▼ AI-Helper

Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

주제어

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가설 설정

25k of PGK1 and TEF1 promoters showed slightly lower expression levels. We hypothesized that Pichia transformants harboring the GAP promoter grew more slowly than transformants with the other promoters due to its higher expression (Fig. 4B & Fig. 5B). The expression level of the lacZ control gene in the pPGKZ0.

제안 방법

PCR cloning of other regions of the PGK1 promoter in various expression vector constructs was accomplished by using other sets of forward and reverse primers, as shown in Table 1. The Pichia TEF-1 promoter region (GenBank Accession No. EFO₁₄948) was PCR amplified from Pichia chromosomal DNA using the following primers; TEF1-F (BgliT) 5'GAA GAT CTA TAA CTG TCG CCT CTT TTA TCT GCC-3, and TEF1- R (T104I) 55-GGT AGG CGC OTT CGA AGT TGG CGA ATA ACT AAA ATG TAT G3';. After double digestion of the PCR product with BgHI and BspT104I, the TFE-1 promoter gene was ligated into the BgHl/BspTlOeI digested pGAPZ-E vector.
The orientation of the inserted PARS1 in recombinant pPGKZ-E (an episomal form of pPGKZ vector) was determined by DNA sequencing analyses. The gene expression level was slightly higher in reverse orientation than in the forward orientation so that only the reverse PARS1 orientation from all the recombinant pPGKZ-E vectors was used in further determinations of gene expression of reporter genes. The recombinant pPGKZ-E vectors harboring various deleted regions of the Pichia PGK1 promoter were electroporated into the X33 strain and Zeocin-resistant transformants were selected and used for a Z)e/(7-galactosidase assay.
To further characterize the Pichia PGK1 promoter and construct a convenient constitutive expression vector for P. pastoris, we deleted the non-essential region of the PGK1 promoter without lowering promoter activity and inserted a Pzc/zzo-specific autonomous replication sequence (PARS1) for episomal expression [4, 8, 9]. We characterized the resultant pPGKZ-E expression vector containing the modified PGK1 promoter by comparing protein production levels achieved with this vector with those of other constitutive promoters.

이론/모형

P-galactosidase activity was determined by the Miller method using O-nitrophenyl glucose as substrate. One galactosidase unit is defined as the amount of enzyme that is able to release 1 μmol O-nitrophenol per min at 37℃ under the assay conditions.

성능/효과

In conclusion, it seems that the modified PGK1 promoter could be an alternative choice for constitutive expression in Pichia when expression of heterologous proteins yields cytotoxic effects to the host strain. Furthermore, because it does not cause an instability in cell growth, the new expression vector can be used for controlled constitutive expression in P.
5). The strong constitutive GAP promoter yielded the highest expression of lacZ reporter gene while 0.25k of PGK1 and TEF1 promoters showed slightly lower expression levels. We hypothesized that Pichia transformants harboring the GAP promoter grew more slowly than transformants with the other promoters due to its higher expression (Fig.

참고문헌 (17)

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