Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli.
The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.
Sharma
Shamik S
SS
Department of Chemical Engineering, 127 Earle Hall, Clemson University, Clemson, SC 29634, USA.
Chong
Shaorong
S
Harcum
Sarah W
SW
D002455:Cell Division / Q000502:physiology; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D047668:Inteins / Q000235:genetics / Q000502:physiology*; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D005346:Fibroblast Growth Factors; C045073:Cre recombinase; D019426:Integrases