Recombination Expression And Activity Research Of Mismatch Binding Protein Muts

Mismatch repair systems exist in a wide range of organisms, due to the existence of this system to ensure that the process of DNA replication fidelity. The prokaryotic E.coli and T.thermophilus both possesee this system,which is found if E.coli first.The mismatch repair protein Muts is one of the mismatch recognition proteins and functions first. Mismatch recognition protein MutS can be individually recognize and bind to unpaired DNA duplexes containing a mismatch, and can be combined with the prominent caused by base deletion or base surplus.Synthetic biology aprpeared in the1960s and formally developing in the21st,has made significant achievements and a series of breakthroughs in the development process. The improvement of DNA automated synthsis efficiency and the expansion of genomic database has developed the DNA systhesis technology, but the genome transplantation technology and large fragments were amplified technology were two synthetic biology technology bottlenecks.DNA mismatch repair is a difficult point for a large fragment amplification technology, mismatch repair mediated by protein is one of the solutions. The protein is a mismatch recognition protein called MutS. This method provides a better solotion of mismatch in the DNA synthesis,so that synthetic biology can achieve better development.In this study, we constructed E.coli and T.thermophilus mismatch recognition protein MutS’s prokaryotic expression vector pET-32(a)-EmutS and pET-32(a+)-TmutS, and express the vector in E.coli BL21(DE3). And get MutS protein in a certain purity throuth the hydrophobic interaction affinity chromatography and ion exchange chromatography.The gel retardation assay was used to detect the mismatch recognition activity of E.coli and T.thermophilus MutS protein, hysteresis band will emerge which contains the mismatch site. When the radio of DNA:E-MutS protein molecular get1:4, after sequencing, the mismatch rate of containing1-3mismatch sites has decrease from 53%、60%、58%to36%、35%、33%. The DNA mismatch rate was reduced by17%,25%and25%. T.thermophilus MutS protein fuctions in the DNA double-stranded containing a wrong e sites DNA mismatch rates decrese from58%、59%、57%to35%、32%、31%,when the radio of DNA:MutS protein molecular were1:4,1:6,1:8, reduced by23%、27%、26%,the double-stranded DNA mismatch rates success to get lower.As a result, we have successfully constructed, expressed and purified the MutS protein of E. coli and T.thermophilus, make the activity research through the gel retardation assay. we concluded that the E-MutS and T-MutS protein could significantly reduce the DNA mismatch rate, and we have found the best ridio of molecule of T-mus functioning in the1/1000of DNA mismatch rate is1:6-1:8. The activity of T-Mus is better than E-mus when the raio of molecule number is the same.