Gene Cloning, Expression And Study Of The Property Of Elastin-like Of Polypeptide

Elastin-like polypeptide (ELP) is a new recombinant protein purification tag. The artificial polypeptides consist of repeats of the pentapeptide (VPGXG)n,X is the guest residue, which can be any amino acid except Pro, the subscripted n describes the number of repeats.ELP undergoes a reversible phase transition at a specific temperature, this temperature is referred to as the inverse transition temperature (Tt).Below Tt, an ELP is highly soluble in aqueous solution. When the temperature is higher than Tt, ELPs become insoluble and form large micron-size aggregates that are visible to the naked eye.The aggregated polypeptide completely dissolves again when the temperature is lowered below Tt.ELP fusion protein retains this behavior when a target protein is fused to the ELP at the gene level.we can isolate a recombinant ELP fusion protein from the protein solution by repeating inverse transition cycling (ITC), the ELP tag can be proteolytically removed, or self-cleave mediated by intein in response to solution condition shift, then we can recover the pure target protein.In this research,ELP consists of repeats of the pentapeptide "VPGXG"(with highly hydrophobic isoleucine at the X position), We recover ELP[I]-30,ELP[I]-40and ELP[I]-50after gene cloning, prokaryotic expression and purification.Then,we examine the transition temperature and the influences of different factors.The results show that the transition temperature of ELP[I]-30,ELP[I]-40and ELP[I]-50is32.5℃,29.5℃and25.5℃, respectively. We can decrease the transition temperature by adding different concentrations of NaCl,(NH4)2SO4and PEG8000.The transition temperature of the ELP[I]-30decreases to15.5℃by1.25mol/L NaCl,and the transition temperature of the ELP[I]-40decreases to11℃by1.25mol/L NaCl,in addition,the transition temperature of the ELP[I]-50decreases to7.5℃by0.4mol/L (NH4)2SO4.From a design perspective, the transition temperature of ELP tag must be low enough such that ITC can be performed at conditions that do not denature the target protein.In addition, if ELP tag is too long,it reduces the proportion of the target protein in ELP fusion protein,and decreases the relative expression yields of the target protein.Due to its small molecular weight and low transition temperature,the ELPs we desined in this research might be extremely promising recombinant protein purification tags.