Optimization of ELP-intein mediated protein purification by salt substitution.
In this paper we discuss improvements to our previously reported ELP-intein purification system described by Banki et al. [M.R. Banki, L. Feng, D.W. Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags, Nat. Methods 2 (2005) 659-661; W.Y. Wu, C. Mee, F. Califano, R. Banki, D.W. Wood, Recombinant protein purification by self-cleaving aggregation tag, Nat. Protoc. 1 (2006) 2257-2262]. This method is based on the selective and reversible precipitation of ELP-tagged target proteins by gentle heating in the presence of high concentrations of sodium chloride. A critical aspect of this system is that the ELP tag is induced to self-cleave by a mild pH shift after purification. An examination of the Hofmeister series of ions suggested that salts other than sodium chloride may be more efficient for ELP precipitation. Specifically, by replacing sodium chloride with ammonium sulfate to induce ELP aggregation, we were able to reduce the required salt concentration by almost 4-fold, and the precipitation steps could be conducted at room temperature instead of 37 degrees C. This results in a cheaper, gentler, and more scaleable purification method. To demonstrate these advantages, green fluorescent protein and beta-lactamase were purified using the newly optimized conditions in side-by-side comparisons to the previous method. The results indicate that both specific activity and yield were improved with the new conditions. These improvements thus significantly increase the attractiveness of this highly general and economical method for recombinant protein purification.
Fong
Baley A
BA
Department of Chemical Engineering, Princeton University, Princeton, NJ 08544, USA.
Wu
Wan-Yi
WY
Wood
David W
DW
D000645:Ammonium Sulfate / Q000737:chemistry; D004549:Elastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D047668:Inteins / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry*; D013696:Temperature; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D012492:Salts; D049452:Green Fluorescent Proteins; D004549:Elastin; D001618:beta-Lactamases; D000645:Ammonium Sulfate