Bacteriophage T7 RNA polymerase-based expression in Pichia pastoris

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• 作者：Birgit Hobl ; Bj?rn Hock ; S ; ra Schneck ; Reinhard Fischer ; Matthias Mack
• 关键词：Recombinant protein production ; Pichia pastoris ; T7 RNA polymerase ; Internal ribosomal entry site ; Pichia pastoris autonomous replicating sequence
• 刊名：Protein Expression and Purification
• 出版年：2013
• 出版时间：November, 2013
• 年：2013
• 卷：92
• 期：1
• 页码：100-104
• 全文大小：615 K

文摘

A novel Pichia pastoris expression vector (pEZT7) for the production of recombinant proteins employing prokaryotic bacteriophage T7 RNA polymerase (T7 RNAP) (EC 2.7.7.6) and the corresponding promoter pT7 was constructed. The gene for T7 RNAP was stably introduced into the P. pastoris chromosome 2 under control of the (endogenous) constitutive P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (pGAP). The gene product T7 RNAP was engineered to contain a nuclear localization signal, which directed recombinant T7 RNAP to the P. pastoris nucleus. To promote translation of uncapped T7 RNAP derived transcripts, the internal ribosomal entry site from hepatitis C virus (HCV-IRES) was inserted directly upstream of the multiple cloning site of pEZT7. A P. pastoris autonomous replicating sequence (PARS1) was integrated into pEZT7 enabling propagation and recovery of plasmids from P. pastoris. Rapid amplification of 5¡ä complementary DNA ends (5¡ä RACE) experiments employing the test plasmid pEZT7-EGFP revealed that transcripts indeed initiated at pT7. HCV-IRES mediated translation of the latter mRNAs, however, was not observed. Surprisingly, HCV-IRES and the reverse complement of PARS1 (PARS1rc) were both found to display significant promoter activity as shown by 5¡ä RACE.