Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag

作者：Qing Zhao, Wanghui Xu, Lei Xing, Zhanglin Lin · 发表于：Microbial Cell Factories · 年份：2016 · DOI：10.1186/s12934-016-0534-3 · 被引用次数：57 · 研究领域：Peptidase Inhibition and Analysis、Chemical Synthesis and Analysis、Monoclonal and Polyclonal Antibodies Research

BACKGROUND: Peptides have recently become attractive for therapeutic applications. However, efficient production of medium- to large-sized peptides (30-100 amino acids [aa]) remains challenging both by recombinant and chemical synthesis. We previously reported the formation of active enzyme aggregates in Escherichia coli cells induced by the short β-structured peptide ELK16 (LELELKLKLELELKLK) and developed a streamlined protein expression and purification approach. In this approach, a cleavable self-aggregating tag (cSAT) consisting of an intein molecule and ELK16 was used to release the recombinant peptides with reasonable purity from active aggregates. RESULTS: In this work, we extended the cSAT approach to a generalized expression and purification solution for a set of medium- to large-sized peptides with important therapeutic uses, including human glucagon-like peptide 1 (31 aa), B-type natriuretic peptide (32 aa), exendin 4 (39 aa), chemokine (C-C motif) ligand 5 (also known as RANTES, 66 aa), stromal cell-derived factor 1α (67 aa), insulin-like growth factor 1 (70 aa), and leptin (146 aa). After intein-mediated cleavage, the soluble peptides were released directly into the supernatant while insoluble peptides could be refolded and purified by reverse phase high-performance liquid chromatography. Additionally, an N-terminal thioredoxin tag was added upstream of the target peptides, which can be removed by enterokinase cleavage, generating native N-terminus for target pep...