Cloning, prokaryotic expression and function of the Bufo bufo gargarizans tryptophan hydroxylase I gene
10.16438/j.0513-4870.2015-1095
- VernacularTitle:中华大蟾蜍色氨酸羟化酶BbgTPH1基因的克隆、原核表达及功能鉴定
- Author:
Yan-fang LIU
1
;
Xi CHEN
1
;
Hong LING
1
;
Dong-chun LIU
1
;
Ming-yu XIA
2
;
Dong WANG
1
Author Information
1. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
2. School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
Bufo bufo gargarizans;
tryptophan hydroxylase I;
cDNA;
prokaryotic expression
- From:
Acta Pharmaceutica Sinica
2016;51(7):1158-
- CountryChina
- Language:Chinese
-
Abstract:
Tryptophan hydroxylase I (TPH1) catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1984 bp (GenBank accession No. JQ768313) with a 1443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 kDa and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L-1 IPTG at 20℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.
