Lennick M, Haynes J R, Shen S H
Molecular Genetics Group, Connaught Research Institute, Willowdale, Ontario, Canada.
Gene. 1987;61(1):103-12. doi: 10.1016/0378-1119(87)90369-6.
A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli. In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone. This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide. The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form.
本文描述了一种方法，该方法可使α-人心房钠尿肽在大肠杆菌中以稳定形式高产合成。在最终的表达系统中，合成的α-hANP基因的八个拷贝串联连接，中间由编码4个氨基酸（aa）连接子的密码子隔开，赖氨酸残基位于28个氨基酸的激素的真实N端和C端两侧。该序列又与一个片段的3'端相连，该片段包含lac启动子和编码β-半乳糖苷酶前七个N端氨基酸的前导序列。表达的多结构域蛋白在细胞内积累形成稳定的包涵体，通过尿素提取不溶性细胞部分可轻松纯化。纯化的蛋白用内肽酶Lys-C消化裂解成单体，用羧肽酶B消化修剪以暴露真实的C端，并用铁氰化钾温和氧化形成单个二硫键。通过反相液相色谱法显示，完全加工的重组肽在还原形式和折叠形式下均与化学合成的标准α-hANP无法区分。
