Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan.
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
Molecules. 2021 Oct 14;26(20):6207. doi: 10.3390/molecules26206207.
The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of gene (8) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of hosts, and XL1-Blue with gene 8 revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.
采用 作为宿主菌，通过重组表达尝试生产由 13 个氨基酸组成的肽类激素 α-促黑素细胞激素（α-MSH）。为实现这一目标，设计了一个包含 8 个串联重复 基因（8）的合成基因，用于核糖体合成 8 个 α-MSH。该策略的优点是可以减少肽对宿主细胞的毒性，并获得更高的产量。在肽之间引入胃蛋白酶切割位点，用于酶解获得单体 α-MSH 肽。构建的质粒被转化到不同的 宿主菌菌株中，基因 8 的 XL1-Blue 显示出最高的 8 α-MSH 产量。尽管细胞裂解后 8 α-MSH 在不溶性沉淀中部分分级，但胃蛋白酶切割能够产生可溶性 α-MSH 肽，如通过质谱分析和肽活性测定所分析和证实的。使用 HPLC 定量测定 α-MSH 的产量，LB 培养物的产量为 42.9mg/L。本研究证明了使用串联重复基因的重组表达生产 α-MSH 的可行性。该生产过程涉及最小的后处理和加工步骤，可以进行放大以用于工业应用。
