Fusion Expression And Purification Of Antihypertensive Active Peptide

Hypertension is a major cause of cardiovascular disease death. Along with rapid economic development and lifestyle change, morbidity rate of hypertension showed a continuously increasing trend. Various drugs have been developed, such as compound antihypertensive tablets, reserpine, and so on. However, it clinically depressed that these drugs have a adverse effects on health, slowing down or speeding up heart rate. Consequently, increasing researchers paied attention on antihypertensive active peptide which is prepared by natural biological macromolecules-protein. The advantages of antihypertensive active peptide are safe non-toxic, no side effect, no effect on normal blood pressure and mild antihypertensive effect. Due to its inhibiting effect on angiotensin converting enzyme(ACE), antihypertensive active peptide can lower blood pressure.The preparation of antihypertensive active peptide is mainly by protease hydrolysis, microorganism fermentation and genetic engineering method. And it has been reported that antihypertensive active peptide can be successfully separated from enzymatic hydrolysates and fermentation products. Due to complicated components of enzymatic hydrolysates and rare fermentative strain applied, development of these two methods are limited in the application of separation and purification of antihypertensive active peptide. Moreover, recent researches show that genetic engineering method has huge development potential on production of antihypertensive active peptide. However, there were some problems in the technical realization and its application was limited in the actual operation. Furthermore, from the perspective of gene expression, on one hand, antihypertensive active peptide sequence was too short to be easily degraded by engineering bacteria protease. Antihypertensive active peptide monomer could not be expressed directly. On the other hand, protein could not correctly fold, leading to the formation of inclusion body and renaturation was time-consuming and laborious. From the perspective of protein purification, cleavage sites usually could not be effectively identified by protease and the tag was difficultly removed by protease digestion. Moreover, affinity purification medium was expensive and the purification steps were complicated. It was high cost, time-consuming steps and to use toxic reagent.On the analysis above, this article was firstly based on restriction sites of gastrointestinal proteases and relationship between structure and property of antihypertensive peptide to collect antihypertensive peptide monomers of high antihypertensive activity in vivo and vitro, 20 kinds of antihypertensive peptide were linked to compose an antihypertensive peptide multimer(AHPM) in tandem way through high release rates active peptide fragments as the link fragments. Optimum gene parameters were critical to the efficiency of gene expression including Escherichia coli codon bias and GC content. And then recombinant strain named as Top10/p UC57-Simple-AHPM was constructed.Secondly, the recombinant plasmid p ET28a-L2(252-273)-SUMO-AHPM was constructed and transform into E. coli BL21(DE3). The gene sequencing result showed that AHPM was accurately linked into carboxyl terminal of L2(252-273)-SUMO. Then the recombinant protein L2(252-273)-SUMO-AHPM was expressed in Escherichia coli BL21(DE3). After OD600 value of bacteria solution was 1.0 at 37 oC and with 0.1 mmol/L isopropyl-D-thiogalactopyranoside at 30 oC, induction for 7 h, the recombinant AHPM fused with L2(252-273)-SUMO tags was successfully expressed and reached the maximal production. In this condition, the recombinant protein L2(252-273)-SUMO-AHPM(27 k Da) expression level in E.coli BL21(DE3) was reached to 254 mg per liter culture medium and soluble form accounted for above 95%.Finally, L2(252-273)-SUMO double fusion tags were used in purifying recombinant AHPM which was expressed in E.coli BL21(DE3). According to the specificity adsorption function between L2(252-273) and diatomite filter aid STD(hereinafter referred to as diatomite), the fusion protein L2(252-273)-SUMO-AHPM was adsorbed to diatomite. And when temperature was 25 oC, oscillating rate was 150 rpm, reacting time was 2.0 h, the adsorption rate was 94.50%. Then by digestion with recombinant SUMO protease(Ulp1-K10), the AHPM was separated by diatomite while L2(252-273)-SUMO tags and Ulp1-K10 were remained on diatomite and 31.68 mg of AHPM was obtained from per liter culture medium. Meanwhile the release of high active antihypertensive peptide from the AHPM was confirmed by the simulated gastrointestinal digestion, with IC50 values of 56.22±1.03 μg/m L and 56.10±1.05 μg/m L, respectively.This study have succeeded in designing, expressing and purifying the high activity AHPM by biological engineering. It provides industrial production of antihypertensive active peptide with a convenient approach and a novel research idea for other bioactive peptide production by genetic engineering.