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为提高重组人心房利钠肽(Atrial natriuretic peptide,ANP)的表达量,将3个ANP通过赖氨酸(Lysine,K)串联,并构建相对应的重组表达载体p ET28a(+)/ANP3。转染大肠杆菌进行诱导表达,目的蛋白约占菌体总蛋白的60%。经过包涵体变复性,赖氨酸酶(Lys-C)和羧肽酶(CPB)水解,以及一系列层析纯化,每升培养液可获得约16 mg的ANP蛋白。最终,纯化后的ANP经UPLC及Tricine SDS-PAGE鉴定,纯度大于90%,LC-MS鉴定显示其分子量为3 080 Da,且为二硫键正确形成的ANP单体,通过ELISA试剂盒检测,其具有和参比品一致活性。本研究为ANP的大规模制备打下了基础。同时,所采用的串联表达技术也为其他多肽类药物的重组表达提供了新的思路。
To improve the expression of recombinant humantrialtrial atriuretic peptide (ANP), three ANPs were ligated in series with Lysine (K) to construct the corresponding recombinant expression vector pET28a (+) / ANP3. E. coli was transfected for expression, the target protein accounted for about 60% of the total bacterial protein. Approximately 16 mg of ANP protein was obtained per liter of culture broth after inclusion body renaturation, hydrolysis of lysine (Lys-C) and carboxypeptidase (CPB), and a series of chromatographic purification. Finally, the purified ANP was identified by UPLC and Tricine SDS-PAGE with a purity greater than 90%. LC-MS analysis showed that the purified ANP had a molecular weight of 3080 Da and was correctly formed by disulfide bond. The purified ANP was detected by ELISA kit, It has the same activity and reference substance. This study laid the foundation for the large-scale preparation of ANP. At the same time, the tandem expression technique adopted also provides a new idea for the recombinant expression of other polypeptide drugs.