Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 79, Issue 1, Pages 72-80Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2011.04.016
Keywords
Inclusion bodies; C5a; CCL18; FGF beta; LIF; Protein expression; Recombinant cationic proteins; Solubility enhancement; Complement; Stem cells
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Funding
- Tobacco Related Disease Research Program [TRDRP-16RT-0134]
- NIH [R21NS062428, R41 CA126004, TRDRP-13RT-0083, R21 HL094878]
- Multiple Sclerosis National Research Institute [4061]
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An expression method has been developed to produce soluble cationic polypeptides in Escherichia coli while avoiding inclusion body deposition. For this technique the recombinant product is linked through a thrombin or factor Xa susceptible bond to the amino-terminal domain of the precursor of eosinophil major basic protein (MBP). This N-terminal domain is strongly acidic and is apparently able to shield eosinophils from the potentially injurious activities of MBP. It was reasoned that constructs of this acidic domain with small heterologous cationic proteins expressed in E. coli could result in soluble expression while preventing trafficking and packaging into insoluble inclusion bodies. This has been demonstrated using four examples: complement C5a, CCL18, fibroblast growth factor-beta, and leukemia inhibitory factor, whose isoelectric points range from 8.93 to 9.59. Further general applicability of this technique has been shown by using two different expression systems, one which encodes an amino-terminal oligo-histidine leash, and another that codes for an amino-terminal glutathione-S-transferase. Thus the utility of coupling MAP to cationic polypeptides for the purpose of soluble heterologous protein expression in E. coli has been demonstrated. (C) 2011 Elsevier Inc. All rights reserved.
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