Using DNA Shuffling Method To Generate Thermosensitive CI Repressor Protein Of λ Phage

CI represser protein plays a key role in controlling the X, phage infection from lysogen to lysis. Several thermo-labile CI variants had been isolated, among which the most famous was CIts857. In genetic engineering, the design of expression vectors based on the strong PL (or PR) Lambda phage and CIts857 was able to control the expression of a cloned gene. Escherichia coli recombinant cultures carrying such vectors are usually induced by a temperature shift from 30℃ to 42℃. It is known that the rate of protein degradation in E.coli cells above 40℃ is significantly increased because the expression of protease is induced by heat-shock. On the other hand, genes controlled by these vector system are ineffectively expressed at temperatures lower than 40℃. A regulatory system for effective expression of genes at temperature of normal physiological condition for bacterial cells(37℃) is desirable. DNA shuffling, which mimicked natural recombination of DNA, was a method for the reassembling of related genes. This technique not only recombined DNA fragment, but also introduced mutations at a low controlled rate. In this study, DNA shuffling method was used for molecular evolution in vitro to generate thermo-sensitive CI represser protein.First, cl gene was cloned from the wild type X, genome using PCR method. After enzyme digestion, they were inserted into the prokaryotic expression pYPX4062 (Genbank No: AY178451). The expression vector designated as pYPX4062-cI after color change detection and sequence verification was constructed. clts857 gene was also cloned from the Xclts857 type phage genome and made pYPX4062-c!ts857 vector as a control at the same time.Second, cl gene was amplified from plasmid pYPX4062-cI. Optimization protocol of DNA shuffling procedure was used to reassemble the cl gene. After restriction enzyme digestion the shuffling DNA, they were inserted into the prokaryotic expression pYPX4062Third, the ligation products were transformed to EG48, a lacZ gene deficiency E.coli strain, by electroporation. A mutant plasmid library containing about 1.04×106 clones was generated. 132 temperature sensitive clones were obtained by screening of 1 × 105 clones. Compared to cIts857, 5 clones were selected. They had clear phenotype and were designated as cIts6, cIts 14, cIts81, cIts85 and cits 107. The galactosidase activity assay results meant the induce effects of these clones under 37 ℃ were 1.38, 3.77, 5.12, 3.35 and 1.61 times of clts857.Finally, DNA sequencing of above 5 clones showed that clts6 had 3 mutations, A77G,G259C, T506C; clts14 had 2 mutations, A199G, A602G; cltsSl had 4 mutations, A44G, G92A, G379A, A629T; clts85 had only 1 mutation, C188T; cIts107 had 3mutations, G160A, A488G, T561C. Among 13 mutations, there was only one synonymous mutation and did not cause amino acid alteration. Every clone had mutation located in N-terminal of cl represser protein. The mutation locus of 199 was the same as cIts857. The mutation locus of 188 was the same as another temperature sensitive strain c!tsU46, but the amino acid was different.Our results show that DNA shuffling, an effective method to generate mutation and recombination can facilitate us to do molecular evolution in vitro. The five thermo-sensitive variants obtained by this study can help us to construct regulatory system for effective expression of genes at temperature of normal physiological condition for bacterial cells and provide us good materials to understand the structure-function relationships of represser proteins.