分子串联重复策略用于制备超短肽

Molecular tandem repeat strategy for production of ultrashort peptides

摘要:

超短肽具有更好的稳定性、组织渗透性、生物相容性以及更低的免疫原性。GHK(glycyl-l-histidyl-l-lysine)和GQPR(glycyl-l-glutamyl-l-prolyl-l-arginine)具有刺激胶原蛋白产生、减缓胶原蛋白降解的作用，作为抗皱成分广泛应用于化妆品。超短肽一般都是通过固相合成方法制备，其缺陷是制备过程中大量使用有机化学试剂而造成环境负担，故本文探讨了一种设计和制备超短肽的新方法。因序列短而无法直接重组表达，文中首先构建了适用于融合表达的载体骨架pET28a-Trxm。以GHK和GQPR串联重复基因作为滚环扩增的基本单元(tandem repeat of short peptides,TRSP)，反应时随机掺入5-甲基胞嘧啶获得长基因片段，然后经Acc65 Ⅰ和Apa Ⅰ消化产生随机长度的基因。胶回收500 bp到1500bp的DNA片段，克隆得到表达载体pET28a-Trxm-(TRSP)_n并转化获得重组菌。双酶切及测序结果表明，成功构建获得串联重复数n=1、2、3、4、6、7、8、9的阳性克隆。蛋白表达结果显示，当串联重复数n=1、2、3、4、8、9时均有相应融合蛋白表达，表达水平随着重复数增加而降低。Trxm-(TRSP)₁表达水平最高，达总蛋白的50%，而Trxm-(TRSP)₂表达水平为总蛋白的30%。进一步地，含Trxm-(TRSP)₁的清液先后经肠激酶和胰蛋白酶切割后，HPLC分析结果表明，成功获得超短肽GHK和GQPR。该结果对于超短肽重组制备的工业化应用具有重要价值。

关键词:串联重复基因;滚环扩增;超短肽;融合蛋白;重组制备

Abstract:

Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)_n. The pET28a-Trxm-(TRSP)_n was introduced into E.coli BL21(DE3) to generate a library of Trxm-(TRSP)_nsequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)₁achieved 50% of the total protein, while the expression level of Trxm-(TRSP)₂ was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)₁ were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.

Key words:tandem repeat gene;rolling circle amplification;ultrashort peptide;fusion protein;recombinant production