一种基于PGK1启动子的毕赤酵母组成型表达系统。

A constitutive expression system for Pichia pastoris based on the PGK1 promoter.

作者信息

Arruda Andrelisse, Reis Viviane Castelo Branco, Batista Vinícius Daniel Ferreira, Daher Bruno Sahim, Piva Luiza Cesca, De Marco Janice Lisboa, de Moraes Lidia Maria Pepe, Torres Fernando Araripe Gonçalves

机构信息

Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70901-900, Brazil.

出版信息

Biotechnol Lett. 2016 Mar;38(3):509-17. doi: 10.1007/s10529-015-2002-2. Epub 2015 Nov 19.

DOI:10.1007/s10529-015-2002-2

PMID:26585331

Abstract

OBJECTIVES

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

RESULTS

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

CONCLUSIONS

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

摘要

目的

基于内源性糖酵解PGK1启动子开发一种用于毕赤酵母组成型表达的新型载体。

结果

携带至少415 bp PGK1启动子序列的毕赤酵母质粒可用于通过在此位点添加来驱动质粒整合，而不影响细胞生长。基于此结果，构建了一种新的毕赤酵母整合载体pPICK2，其具有一些有助于在该酵母中生产蛋白质的特征：一个约620 bp的PGK1启动子片段，在酵母转化前有三种用于质粒线性化的限制性酶切位点选择；一个密码子优化的α因子分泌信号、一个新的多克隆位点，以及用于载体在细菌中繁殖和酵母转化子筛选的kan标记。