The New Type Of Recombinant Pisum Sativum Prophobilinogen Deaminase And Site-directed Mutagenesis

The common pathway of all tetrapyrroles biosynthesis was catalyzed by three enzymes:5-amniolevulinate dehydrates (ALAD), prophobilinogen deaminase (PBGD) and uroporphyinogn III synthase (UROS). Porphobilinogen is one of regulating enzymes in the common pathway . To research high efficient expression of Pysium Sativum PBGD in E.coli, The dual-tag pQE30sPBGD with T5 promotor and the dual-tag p28PBGD were constructed. To make sure the contribution of PBGD Ala188 to enzyme activity, the site Ala188 was mutagenesized as Ser188 by site-directed mutagenesis. To avoid damnification of host cell from accumulation of the product UroI, UROS encoding B.Substilis hemD vector was co-expressed with p28PBGD. To regulate the expression of recombinant PBGD, ALAD encoding B.Substilis hemB was co-expressed with p28PBGD. The co-expression vectors p28PBGD /pA-ALAD and p28PBGD/pA-UROS was induced and then purified by Ni-NTA affinity chromatography. The results were as follows: 1 The T5 promotor containing vector pQE30sPBGD and T7 promotor containing vector p28PBGD were consturced. The recombinant PBGD was expressed with N-terminus His6 and C-terminus strep-tag. 2 The conservative amino acid A188S of pea PBGD was mutagenesized by site directed mutagenesis. 3 The upstream enzyme and the downstream enzyme, ALAD and UROS were inserted into pET-3a. Then the T7 promotor and RBS carrying gene fragments of the two enzymes were incised by restriction endonucleases, were inserted into pCYC184.. 4 The p28PBGD was co-transferred with pA-ALAD and pA-UROS respectively. 5 The co-expression vector p28PBGD/ pA-ALAD and p28PBGD/ pA-UROS were induced and purified by Ni-NTA affinity chromatography. The results of SDS-PAGE indicated that there was a main belt from purification and molecular weight was about 40KD.