Dataset Information

Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag.

ABSTRACT: The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggregating peptide ELK16. In this scheme, the target proteins were fused in the C-termini with cSAT and expressed as insoluble aggregates. After intein self-cleavage, target proteins were released into the soluble fraction with high yield and reasonable purity. We demonstrated the applicability of this scheme by preparing seven model viral peptides, with lengths ranging from 32 aa to 72 aa. By adding an N-terminal thioredoxin tag, we enhanced the yield of target peptides released from the aggregates. The purified viral peptides demonstrated high antigenic activities in ELISA and were successfully applied to dissecting the antigenic regions of influenza hemagglutinin. The cSAT scheme described here allows for the rapid and low-cost preparation of multiple antigenic peptides for immunological screening of a broad range of viral antigens.

SUBMITTER: Xu W

PROVIDER: S-EPMC5093863 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag.

Xu Wanghui W Zhao Qing Q Xing Lei L Lin Zhanglin Z

Scientific reports 20161103

The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggr ...[more]

PMID: 27808126

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Project description:BackgroundPeptides 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.ResultsIn 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.ConclusionsThe 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.

Dataset Information

Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag.

Publications

Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag.

Similar Datasets

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