摘要
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。
In order to improve the expression of recombinant human atrial natriuretic peptide（ANP）, a new plasmid（p ET28a（＋）/ANP3） containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21（DE3） and induced by IPTG, about 60% of the total-cell-protein was the target protein, His6-ANP3. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B（CPB） and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.
出处
《生物工程学报》
CAS
CSCD
北大核心
2016年第9期1273-1285,共13页
Chinese Journal of Biotechnology
基金
国家高技术研究发展计划(863计划)(No.2014AA021004)
国家新药创制重大专项(Nos.2013ZX09101021
2013ZX09401303)
上海市重点实验室(Nos.13DZ1930100
14DZ2272300)
上海市优秀学术带头人(No.13XD1424000)
上海市生物医药领域科技支撑项目(No.15431906100)
上海市重点学科基金(No.B905)资助~~
关键词
心房利钠肽
串联表达
多肽纯化
atrial natriuretic peptide
tandem expression
peptides purification
