title: Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag.
abstract: 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 peptides. Final yields of the peptides ranged from 0.1 to 1.8 μg/mg wet cell weight at laboratory scale.
  
  
  CONCLUSIONS
  The approach described in this study provides a fast and efficient route to express and purify peptides that are difficult or expensive to produce by chemical synthesis or by ordinary recombinant methods. It is particularly well suited for large peptides, peptides likely to be degraded, and peptides that have toxic effects on the host. It can greatly reduce the cost and time of downstream processing, and thus may be useful for both industrial manufacture and laboratory applications.
authors: Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
authors: Qing
authors: Q
authors: Zhao
authors: Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.|, Novozymes, China Headquarters, 14 Xinxi Road, Shangdi Zone, Haidian District, Beijing, 100085, China.
authors: Wanghui
authors: W
authors: Xu
authors: Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.|China National Petroleum & Chemical Planning Institute, 16th Floor, 7 Block, Hepingli Zone, Beijing, 100013, China.
authors: Lei
authors: L
authors: Xing
authors: Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China. zhanglinlin@mail.tsinghua.edu.cn.
authors: Zhanglin
authors: Z
authors: Lin
mesh_terms: D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods*; D047668:Inteins; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D066329:Protein Aggregates; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D010455:Peptides; D066329:Protein Aggregates; D011994:Recombinant Proteins
keywords: Authentic N-terminus; Cleavable self-aggregating tag; Therapeutic peptide production; Thioredoxin fusion tag