Construction, Expression And Purification Of Prokaryotic Vector Of AIF-1

Allograft inflammatory factor 1 (AIF-1)/ Daintain is a 147-aa residue polypeptide with a mass of 17kDa,characterd as a macrophage and T Lymphocyte factor by several European and American searth teams in the midanaphase of 90's last century for different aims . Daintain/AIF-1 has an Ca2+-binding EF-hand suggested some function in Ca2+ metabolism,and an internal 44-residue segment with the sequence pattern–KR–KK–GKR–, a motif typical of peptide hormone precursors, regulating insulin secretion. Daintain/AIF-1 has been verified an important protein in tumor progress, heart/liver transplant rejection, central lesion, and autoimmune disease etc. For interesting structure and function, Daintain/AIF-1 is widespread investigated in biology and medical science.Daintain/AIF-1 is too little to purify from wild material. So its oligo-nucleotide fragment was artificially synthetic based on the amino-acid sequence of Daintain/AIF-1 . After extracting plasmid, enzyme hydrolysis,the fragment was cloned into prokaryotic expression vector pET32-a(+). The recombinant expression plasmid pET32-a(+) -Daintain/AIF-1-6His was transformed into E.coli BL21(DE3). Then the conditions of inducement were optimized at 30℃, 2h. The fusion protein Daintain/AIF-1-6His was solubly expressed. The fusion protein was purified by Sephrose-NI affinity column. The molecular mass of the fraction determined by SDS-PAGE was about 18kDa, Existence of Daintain/AIF-1 was further identified by Western blotting .So the Daintain/AIF-1-6His fusion protein was successfully expressed in E. coli.The work provides preparation for further study of the function of Daintain/AIF-1 and producing the protein with gene engineering for clinical application.