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Polyethyleneimine‐Mediated Chemical Extraction of Cytoplasmic His‐Tagged Inclusion Body Proteins from Escherichia coli
Author(s) -
Nian Rui,
Tan Lihan,
Choe WooSeok
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp070304q
Subject(s) - extraction (chemistry) , protein purification , escherichia coli , chromatography , chemistry , dna , recombinant dna , spermine , solubility , dna extraction , inclusion bodies , biochemistry , enzyme , organic chemistry , gene , polymerase chain reaction
The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His‐tagged model proteins: gloshedobin, a thrombin‐like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD 600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine‐induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost‐prohibitive spermine and EDTA. The residual PEI in the post‐extraction mixture was efficiently counteracted by addition of Mg 2+ , allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI‐mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.