Purification of an elastin-like fusion protein by microfiltration.
This article describes a simple and potentially scalable microfiltration method for purification of recombinant proteins. This method is based on the fact that when an elastin-like polypeptide (ELP) is fused to a target protein, the inverse phase transition behavior of the ELP tag is imparted to the fusion protein. Triggering the phase transition of a solution of the ELP fusion protein by an increase in temperature, or isothermally by an increase in salt concentration, results in the formation of micron-sized aggregates of the ELP fusion protein. In this article, it is shown that these aggregates are efficiently retained by a microfiltration membrane, while contaminating E. coli proteins passed through the membrane upon washing. Upon reversing the phase transition by flow of Milli-Q water, soluble, pure, and functionally active protein is eluted from the membrane. Proof-of principle of this approach was demonstrated by purifying a fusion of thioredoxin with ELP (Trx-ELP) with greater than 95% recovery of protein and with greater than 95% purity (as estimated from SDS-PAGE gels). The simplicity of this method is demonstrated for laboratory scale purification by purifying Trx-ELP from cell lysate using a syringe and a disposable microfiltration cartridge. The potential scalability of this purification as an automated, continuous industrial-scale process is also demonstrated using a continuous stirred cell equipped with a microfiltration membrane.
Ge
Xin
X
Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ont., Canada L8S 4L7.
Trabbic-Carlson
Kimberly
K
Chilkoti
Ashutosh
A
Filipe
Carlos D M
CD
D001331:Automation; D004549:Elastin / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005374:Filtration / Q000379:methods; D007218:Industrial Microbiology / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D004549:Elastin