Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology.
The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.
Department of Biotechnology, Royal Institute of Technology (KTH), SCFAB, S-106 91, Stockholm, Sweden.
Torbjörn
T
Gräslund
My
M
Hedhammar
Mathias
M
Uhlén
Per Ake
PA
Nygren
Sophia
S
Hober
D000327:Adsorption; D019677:Artificial Gene Fusion; D001483:Base Sequence; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D011499:Protein Processing, Post-Translational
D002411:Cation Exchange Resins; D017931:DNA Primers