Intein-mediated rapid purification of recombinant maxadilan and M65 and their acute effects on plasma glucose.
Maxadilan is a potent vasodilatory peptide present in the salivary glands of the sand fly. Maxadilan and M65, a deletion variation of maxadilan, are agonist- and antagonist-specific for the PAC1 receptor. In order to obtain the recombinant maxadilan and M65 efficiently by intein-mediated single column purification, the genes encoding maxadilan and M65 were designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant maxadilan and M65 with homogeneity over 95% were released from the chitin-bound intein tag by beta-mercaptoethanol. Intraperitoneal injection of the recombinant maxadilan caused an acute elevation of plasma glucose, imitating pituitary adenylate cyclase-activating polypeptide (PACAP) 27, in NIH mice, while the VPAC1-agonist and VPAC2-agonist had no significant effects on the levels of plasma glucose. M65 alone had no effect on the plasma glucose, but blocked the glucose excursion caused by maxadilan by 12.7% and blocked the glucose excursion caused by the PACAP 27 by 11.6%. The acute effects of the recombinant maxadilan and M65 on the plasma glucose indicated that they had the characteristics as the agonist and antagonist for PAC1.
Yu
Rongjie
R
Bio-engineering Institute of Jinan University, Jinan University, Guangzhou, China. rongjie_yu1123@163.com
Yi
Tianhong
T
Zhang
Ling
L
Hong
An
A
Dai
Yun
Y
Zhou
Tianhong
T
D000818:Animals; D001786:Blood Glucose / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D047668:Inteins / Q000502:physiology*; D013058:Mass Spectrometry; D008623:Mercaptoethanol / Q000737:chemistry; D051379:Mice; D008970:Molecular Weight; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D001786:Blood Glucose; D019476:Insect Proteins; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011994:Recombinant Proteins; C069147:maxadilan protein, insect; D008623:Mercaptoethanol