Design and expression of recombinant antihypertensive peptide multimer gene in Escherichia coli BL21.
The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation exchange chromatography under denaturing conditions, followed by refolding together with size exclusion chromatography and gradual dialysis. The resulting yield of the soluble GST-AHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.
Rao
Shengqi
S
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Su
Yujie
Y
Li
Junhua
J
Xu
Zhenzhen
Z
Yang
Yanjun
Y
D000959:Antihypertensive Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004925:Escherichia / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006973:Hypertension / Q000378:metabolism / Q000517:prevention & control; D002479:Inclusion Bodies / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D000959:Antihypertensive Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase; D010447:Peptide Hydrolases