Automated purification of His(6)-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis

In the course of site-directed mutagenesis or directed evolution experiment s, large numbers of protein variants are often generated. To characterize f unctional properties of individual mutant proteins in vitro, a rapid and re liable protein purification system is required. We have developed an automa ted method for the parallel purification of 96 different protein variants t hat takes about two hours. Using a 96-well format, the whole precess can be performed automatically by a pipetting robot. Coupled with a suitable assa y, again using a 96-well format, all variants can be functionally character ized within a few hours. The protein purification procedure described here is based on the interaction between His(6)-tagged proteins and Ni-NTA-coate d microplates. Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we h ave purified and characterized variants of the restriction endonuclease Eco RV, which were produced in an effort to enhance the selectivity of this enz yme. For this purpose, three amino acid residues were randomized in a regio n known from the co-crystal structure to be located at the protein-DNA inte rface. From a library of about 1200 variants, predominantly single and doub le mutants, more than 1000 variants were purified and characterized in para llel, which corresponds to an almost complete screening of the library.