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Protein Expr Purif. 2014 Jun;98:18-24. doi: 10.1016/j.pep.2014.02.016. Epub 2014 Mar 7.

Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.

Author information

1
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
2
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China. Electronic address: sunh@yzu.edu.cn.

Abstract

Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human β defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

KEYWORDS:

Elastin-like peptide; Fusion system; Protein purification; Self-processing module

PMID:
24607361
DOI:
10.1016/j.pep.2014.02.016
[Indexed for MEDLINE]
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