DNA Shuffling And Screening For Obtaining Lactose Super-Repressor In Escherichia Coli

Escherichia coli lactose represser/operator was widely received as a paradigm for understanding gene regulation and protein-DNA interaction. The tac promoter (Ptac) is now one of the most popular promoters used for gene expression. The Ptac-based expression system was regulated by the lac represser protein which is a product of lad gene. The wild-type represser protein can incompletely suppress foreign gene expression which show leaky nature. The leaky expression of toxic gene products is detrimental to host strain or even lethal, so tight repression of expression is desirable or necessary. In this paper, DNA shuffling ,which involves Preparation of the wild-type lad gene, DNase I digestion, Primerless PCR, and Primer PCR, was used to generate mutant lad gene libraries from which it is screened for obtained mutant foe/gene encoding lactose super-repressor.Based on pYPX4062( GenBank No, AY178451 ), we construct prokaryotic expression vector pYF6683. pYPX4062 which contains two expression units is a prokaryotic broad-host-range vector with two kinds of selective marker(Ampr and Kmr). One expression unit is lacZ gene expression unit under the control of PL promoter, the other unit is Gnf gene expression unit under control of GmrP promoter. The terminator of both units is T1T2 coming from X-phage. Compared with pYPX4062, Ptac takes the place of PL and lacZ gene replace Gnf gene in pYF6683. The mutant lad gene is ligated to prokaryotic expression vector pYF6683 digested with BamHI and Sacl. A mutant lad expression library with 5~8×106 transformants has been constructed in prokaryotic expression vector containing lacZ gene under the control of the tac promoter. The four mutant lad genes encoding lactose super-repressor protein have been obtained through blue-white screening with X-gal which serves as enzyme substrate. Lac super-repressor protein can tightly suppress the expression of lacZ gene in the absence of inducer (IPTG) and lacZ gene was highly expressed in the presence of inducer (IPTG).Sequencing and sequence analyses revealed bases change and deduced amino acid substitution in the mutant lad genes encoding lactose super-repressor protein.Amino acid mutant site A43V locates in N-terminal headpiece of represser protein which binds specifically to DNAand amino acids mutant sites S93L, S102N, G315S, Q180R, M2491, I283T all locate in the core region of represser protein which binds to IPTG. Compared with the previous reported lad gene mutant site, A43V and S93L are new identified IPTG-inducible mutants. In addition, amino acids mutant sites S102N, G315S, Q180R, M249K I283T lies in the surface of represser protein crystal structure. It inferred that amino acids changes bring about the conformation change of the repression protein, which results in super-repression.