Engels J W, Glauder J, Müllner H, Tripier D, Uhlmann E, Wetekam W
Universität Frankfurt, Institut für Organische Chemie, Frankfurt/Main, FRG.
Protein Eng. 1987 Jun;1(3):195-9. doi: 10.1093/protein/1.3.195.
By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.
通过化学酶法合成，构建了人生长激素释放激素 - Gly45的（Leu27）类似物[(Leu27)GHRH - Gly45]的基因，并在大肠杆菌中作为与β - 半乳糖苷酶的融合蛋白进行克隆和表达，该融合蛋白受lac启动子和操纵子控制。用异丙基 - D - 硫代 - β - 半乳糖吡喃糖苷诱导后，融合蛋白积累量达到总细胞蛋白的15% - 20%。融合蛋白经溴化氰裂解后，前体肽(Leu27)hGHRH - Gly45通过萃取分离，再经离子交换和高效液相色谱 - RP18色谱法纯化。对纯化后的肽进行测序、等电聚焦、氨基酸分析以及V8蛋白酶消化后的氨基酸分析。随后，通过从新鲜牛垂体中分离并鉴定的肽基甘氨酸α - 酰胺化单加氧酶（PAM）将羧基末端的甘氨酸酰胺化。通过与真实的亮氨酸酰胺参照物进行肽测序，验证了倒数第二个氨基酸亮氨酸的正确酰胺化。
