Expression,Isolation And Purification Of Peptide,GHK,as Tandem Repeats In Escherichia Coli

The glycyl-histidyl-lysine-copper(II)complex(GHK-Cu)is a tripeptide-copper complex.Tripeptide GHK,composed of glycine,histidine and lysine,has a high affinity with copper(II),and can spontaneously form a complex of GHK-Cu.Up to now,GHK-Cu has been proved by many studies to have a variety of functions,including helping DNA repair,anti-inflammation,anti-aging,wound repair and so on.At present,the tripeptide GHK is produced by chemical synthesis,which has a large number of organic solvents are used in the production process,leading to an unfriendly result for the health of operators and for the environment.It is very economical and convenient to produce protein by genetic engineering technology.But for small molecular polypeptides,the low expression and easy degradation are a huge problem.In this study,recombinant tandem GHK protein was expressed in Escherichia coli,and digested by enzyme to produce GHK tripeptide monomer in large quantities.The increase of the copy number of GHK was achieved by utilizing the characteristic of Type-IIS restriction endonuclease cutting arbitrary DNA sequences at a specific position downstream of the recognition site.rp UC19-[GHK]₃₁ with 31 copies of GHK was successfully constructed based on the plasmid p UC19,and the copy number of GHK could be further increased by enzyme digestion and ligation.Furthermore,the expression vector p ET30a(+)-[GHK]₃₁ was constructed,It was transferred into E.coli BL21(DE3),and the recombinant protein[GHK]₃₁ was successfully expressed in E.coli by IPTG induction.The expression amount of[GHK]₃₁ protein accounted for about15%of the total cell protein,up to 33.5 mg/L culture,which solved the problem of low expression of short peptide expressed by genetic engineering.In order to isolate and purify the recombinant protein[GHK]₃₁,we tried the methods of ion exchange chromatography,affinity chromatography,salting-out purification,and combined with the methods of gel filtration,heat treatment precipitation,and isoelectric point precipitation for desalination.Finally,a purification desalination scheme of salting-out-isoelectric point-resalting-out precipitation desalination was established.The purified recombinant protein[GHK]₃₁ was greater than 99.5%,the yield was 35.9%,and the GHK was 12.0 mg/L culture.The purified recombinant protein[GHK]₃₁ was digested by trypsin to release the GHK monomer,and finally a GHK tripeptide with a purity of 98%and a yield of 6.2 mg/L culture was obtained.The target protein could be separated and purified efficiently without the purification column,which reduces the production cost of GHK produced by genetic engineering method and the burden of chemical synthesis on the environment.