The Preliminary Study Of Five Mutants Of An Analgesic-antitumor Peptide From Scorpion Buthus Martensii Karsch

The analgesic-antitumor peptide from Buthus martensii Karsch(BmK-AGAP)is a kind of scorpion venom,which has the activity of analgesia and antitumor.The full length of the sequence of BmK-AGAP is 302bp,which encode 85 amino acid residues.A signal peptide in N-terminal which is made of 19 amino acid residues is linked to AGAP,a mature peptide containing 66 amino acid residues..Although scorpion venom has been researched for several decades,the investigation about the relationship between its structure and function is just beginning.The primary structure of BmK-AGAP was mutated to study the releationship between its structure and function.Firstly,the CDS of BmK-AGAP-K-K was inserted into pET-28a(+) vector,and the recombinant plasmid pET-28a-His-AGAP-K-K was then transformed into E. coli BL21(DE3)and induced by IPTG.to expression.The result showed that the expression of His-AGAP-K-K,mainly existed as inclusive body,was about 32%compare to the total bacterial protein.Secondly,different AGAP genes were obtained by PCR using different lower primers. The C-terminal of the original BmK-AGAP contains two Gly,while the mutants contain two more Lys residues,one more Glu residue and two more Glu residues at the end of the sequence,respectively.In addition,we also obtained two more mutants,K62G and K62E. Then five recombinant plasmids were obtained by inserting those five mutated BmK-AGAP genes into pSYPU-1b vectors,which were pSYPU-lb-His-AGAP-K-K, pSYPU-1b-His-AGAP-E,pSYPU-1b-His-AGAP-E-E,pSYPU-1b-His-AGAP-62-Gly and pSYPU-1b-His-AGAP-62-Glu.After they were transformed into the E.coli BL21(DE3)and induced by IPTG,those five target proteins were all expressed in soluble form.Then the five target proteins were purified from the supernatant of E.coli broth by metal chelating affinity chromatography.And the analgesic activity of them was detected by a animal model of twisting action of the mice induced by acetic acid.Pharmaceutical tests showed that the recombinant AGAPs have the analgesic activity on mice.When giving same dosage(0.1364μmol/kg)of the different proteins(His-AGAP-C-2,His-AGAP-K-K, His-AGAP-E,His-AGAP-E-E,His-AGAP-62-Gly and His-AGAP-62-Glu),the rates of inhibition of twisting action of the mice were 29.36%,51.23%,29.54%,28.76%,22.59%and 14.04%respectively.The relationship between structure and function of AGAP was further explored.by studying of the activity of those five mutants of BmK-AGAP.