Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification

被引：19

作者：

Gordeeva, Tatiana L. ^{[1
]}

Borschevskaya, Larisa N. ^{[1
]}

Sineoky, Sergei P. ^{[1
]}

机构：

[1] VKPM, State Res Inst Genet & Select Ind Microorganisms, Moscow 117545, Russia

来源：

JOURNAL OF MICROBIOLOGICAL METHODS | 2010年 / 81卷 / 02期

关键词：

Gene synthesis; Codon modification; Phytase; HIGH-LEVEL EXPRESSION; PICHIA-PASTORIS; HIGH-FIDELITY; DNA-SYNTHESIS; SEQUENCES; OLIGONUCLEOTIDES; MUTAGENESIS; DESIGN; YEAST;

D O I：

10.1016/j.mimet.2010.02.013

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IFS) method consists of two steps. The first one is the synthesis of 300-400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR). (C) 2010 Elsevier B.V. All rights reserved.

引用

页码：147 / 152

页数：6

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