Abstract: This study is to analyze the codon usage bias of six protein-coding sequences in the genomes of phage TSP4, Thermus thermophilus HB27 and Escherichia coli ,and to investigate the effects of codon bias on the heterologous expression of TSP4 genes.Biological software Editseq and algorithm of RSCU were used to analyze the frequency of synonymous codon usage of TSP4 genes to obtain an index of codon usage bias,which was further compared with that of Thermus HB27 and E.coli homologous genes.The results showed that,based on these compared sequences,TSP4 shared 85% similarity of codon usage frequency with Thermus HB27 but only 65% with E.coli .Higher similarity between phage TSP4 and HB27 indicated that TSP4 might possess an adaptation mechanism at the genetic level for its host HB27 during evolution.To test the effects of codon usage bias on the heterologous expression of TSP4 genes,a potential chaperone-coding gene of TSP4 was used for expression in E.coli BL21 and Rosetta (contains six codons rarely used in BL21 strain),respectively.The results showed that protein expression of the target gene was not detected in E.col i BL21 but was observed at a high level in E.coli Rosetta,indicating that the E.coli Rosetta containing six rare codons contributed significantly to the expression of TSP4 chaperone gene, and codon usage bias had a significant effect on gene heterolougs expression.