Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification.
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 (IPS) method consists of two steps. The first one is the synthesis of 300-400bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15bp 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 1256bp 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).
State Research Institute of Genetics and Selection of Industrial Microorganisms, VKPM, 1-st Dorozhnii proezd 1, Moscow 117545, Russian Federation. tatiana.gordeeva@mail.ru
Tatiana L
TL
Gordeeva
Larisa N
LN
Borschevskaya
Sergei P
SP
Sineoky
D010832:6-Phytase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D010832:6-Phytase