The Study Of The Disulfide Pairs And The Bioactivities Of Recombinant Keratinocyte Growth Factor Type I N-terminal Deleted Mutant

Objective: To study the disulfide pair and the bioactivity of Recombinant keratinocyte growth factor Type I N-terminal first 23 Amino Acids deleted mutant (K23、K25、K102和K106) expressed in E.coli. Methods:(1) Recombinant keratinocyte growth factor Type I N-terminal first 23 Amino Acids deleted mutants(K23、K25、K102和K106)were constructed; (2) The SP sepharoseF.F和Heparin-CL-6BF.F were utilized to get the pure recombinant proteins(K23、K25、K102和K106)(3)The peptide mass mapping of mutants(K23、K25、K102和K106)digested by trypsin was measured by LC-MS; The sites of disulfide bonds were confirmed by specific relative molecular mass variations comparing to peptides reduced by TCEP and unreduced. (4)NBL-7 cell line、NIH3T3 cell line and CCl4 induced acute liver injuring in mouse were utilized to analyse the bioactivities of mutants(K23、K25、K102和K106)in vitro and in vivo. Results: The bioactivity and the stability of the K23 is the most high and the K25 is the lowest. Conclusion:①The KGF with Cys102-Cys106 disulfide bond showed more higher stability;②The deletion or mutation of the Cys106 would be detrimental.