A Chimera Polypeptide with Active Sites of HWTX-I and AAI.
The functional amino acid sequence and its neighbouring fragments in the molecule of Amaranth alpha-amylase inhibitor isolated from seeds of the Mexican crop plant Amaranthus hypochondriacus were transferred, by solid phase chemical synthesis, into N-terminal region of the huwentoxin-I(HWTX-I). The synthetic chimera polypeptide was confirmed by Edman degradation and MALDI-TOF mass spectroscopy. The formation of three disulfide bonds and special conformation of the synthetic chimera was induced by the addition of glutathione. Renatured chimera polypeptide was purified by ion-exchange and reversed phase HPLC. The results showed that the engineered chimera polypeptide exerted obvious inhibitory activity to alpha-amylase from digestive tube of the roach (Periplaneta Americana) at pH 5.5 with the concentration of 9.5x10(-5) mol/L, and also exerted 36% of the neurotoxic activity of the natural huwentoxin-I as shown by the experiments of blockage of the neuromuscular transmission of isolated mouse phrenic nerve-diaphragm preparations. The experiments demonstrated that the structural motif of HWTX-I is well promising for protein engineering, and the solid-phase peptide synthesis is adequate rapid for the engineering of artificially designed small proteins.
Wang
Xian-Chun
XC
College of Life Science, Hunan Normal University, Changsha 410081, China. liangsp@public.cs.hn.cn
Liang
Song-Ping
SP
Luo
Ze-Min
ZM