Structure-function Relationships Of The Analgesic-antitumor Peptide From Scorpion Buthus Martensii Karsch

The analgesic-antitumor peptide（BmK-AGAP）from Buthus martensii Karsch is a venom, that has the character of analgesia and antitumor. AGAP is 198bp long and encode 66 amino acid residues.We successfully changed BmK-AGAP gene sequence to research the releationship between its structure and function. Firstly, Cloned BmK-AGAP gene into expression vector pET-28a（+）, Transformed the recombinant expression vector pET-28a-Tag_{（His）}-AGAP into the E. coli BL21（DE3）, and then induced the expression vector by adding IPTG.. The results showed that the production made up to 35％of the total bacterial protein, while it mainly existed as inclusive body.Secondly, Obtained BmK-AGAP mutations by designing the reverse primer and PCR method. The AGAP gene 3’ has six nucleotides which encode two glycines. One mutation’s 3’ has three nucleotides encoding one glycine, and another mutation’s 3’ has no nucleotides encoding glycine. Cloned the BmK-AGAP and two mutations into expression vector pSYPU-1b. Transformed the recombinant expression vector pSYPU-1b-Tag_{（His）}-AGAP-C-2、pSYPU-1b-Tag_{（His）}-AGAP-C-1 and pSYPU-1b-Tag_{（His）}-AGAP-C-O into the E. coli BL21（DE3）, and then induced the expression vector by adding IPTG. The three proteins were soluble.Finally, Purified the different proteins by means of metal chelating chromatography and ion exchanged chromatography. We use the animal model of twisting action of the mice induced by acetic acid to detect analgesic activity. Pharmaceutical tests showed that the recombinant AGAP has analgesic on mice. At the same dosage of 0.1364μmol/kg body weight, Tag_{（His）}-AGAP-C-2、Tag_{（His）}-AGAP-C-1 and Tag_{（His）A}GAP-C-0 peptide has the rate of inhibition 28.76％（p＜0.01）、62.33％（p＜0.01）、81.74％（p＜0.01）respectively. It showed that the analgesic activity of BmK-AGAP increased when its C-terminus amino acids is removed one by one.