Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli.
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 OD600 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 Mg2+, 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.
Nian
Rui
R
Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore.
Tan
Lihan
L
Choe
Woo-Seok
WS
D001426:Bacterial Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D005285:Fermentation; D006639:Histidine / Q000737:chemistry*; D002479:Inclusion Bodies / Q000737:chemistry*; D007202:Indicators and Reagents; D008670:Metals / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016324:Sequence Tagged Sites
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007202:Indicators and Reagents; D008670:Metals; D011994:Recombinant Proteins; D006639:Histidine; D011094:Polyethyleneimine