大肠杆菌holo-ACP突变体的表达纯化及相应acyl-ACP的性质初探

Expression and Purification of holo-ACP Mutant and Property Study on the Corresponding acyl-ACP from E`scherichia Coli

酰基酰基载体蛋白(acyl-acyl carrier protein,acyl-ACP)作为酰基供体,在脂肪酸、聚酮等天然产物合成途径中具有重要作用.目前,常以酰基-辅酶A(acyl-CoA)来代替尚未商品化的acyl-ACP进行相关酶的体外活性研究.由于底物蛋白acyl-ACP的ACP部分与相关酶发生相互作用,影响酶的催化特性,这种替代无法真实认识相关酶的酰基转移特性.本文以大肠杆菌pET-28a(+)-ACP为模板,构建12株单点突变体,表达纯化出相应holo-ACP,并进一步合成C16:0-ACP和C18:1-ACP.高效液相色谱的结果表明,ACP单点突变会影响acyl-ACP整体的性质,其中T40残基的改变对acyl-ACP的疏水性、紫外吸收响应和稳定性均产生了显著影响.ACP突变体的性质表征为acyl-ACP与相关酶的互作机制研究奠定了基础.

As an acyl donor,the acyl-acyl carrier protein(acyl-ACP)plays pivotal roles in biosynthesis of various natural products,including fatty acids,polyketones and so on.At present the acyl-CoA has been used as a substitute for uncommercialized acyl-ACP to perform in vitro study of the relevant enzyme activity.By using acyl-CoA,the catalytic specificity of the enzymes can't be revealed correctly because the substrate protein acyl-ACP could interact with the relevant enzymes through the ACP.Based on the plasmid pET-28a(+)-ACP derived from Escherichia coli,12 single-site ACP mutants were designed,and the corresponding holo-ACPs were expressed and purified.These holo-ACPs were subsequently used as substrates to synthesize C16:0-ACP and C18:1-ACP.The HPLC results show that,ACP mutant with change in a single amino acid can effect on the acyl-ACP feature.In particular,the ACP mutant with change in T40 residue has notable impacts on hydrophobicity,ultraviolet absorption response and stability of acyl-ACP as well.