Automated Purification of Hi s 6 -Tagged Protein s Allows Exhaustive Screening of Librarie s Generated by Random Mutagenesi s

In the course of site-directed mutagen e sis or directed evolution experiments, larg e numbers of protein variants are often gene r ated. To characterize functional propertie s of individual mutant proteins in vitro, a rapid and reliable protein purification sy s tem is required. We have developed an auto mated method for the parallel purification of 96 different protein variants that take s about two hours. Using a 96-well format , the whole process can be performed auto matically by a pipetting robot. Coupled with a suitable assay, again using a 96-well fo r mat, all variants can be functionally cha r acterized within a few hours. The protein purification procedure described here i s based on the interaction between Hi s 6 tagged proteins and Ni-NTA-coated m i croplates. Typical yields are 3–8 pmol pur i fied protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and cha r acterized variants of the restriction endonu clease Eco RV, which were produced in an effort to enhance the selectivity of this en zyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to b e located at the protein-DNA interface. From a library of about 1200 variants, predom i nantly single and double mutants, mor e than 1000 variants were purified and cha r acterized in parallel, which corresponds to an almost complete screening of the library .