Site-directed mutagenesis (SDM) is a powerful tool for analyze protein structure and function, protein folding, and enzyme mechanism (). Several protocols for SDM by polymerase chain reaction (PCR) are published (, , , , , , ). Here, two protocols, which turned out to be robust and efficient in the authors' hands, are described in detail. Both methods comprise two steps: In the first step the desired mutation is introduced by a PCR primer used to amplify one part of the target gene. In a second step, this PCR product is then used as a megaprimer to amplify the full gene containing the mutation. Both methods deviate in how the mutated gene is introduced back into its cloning vector: In method 1, the whole vector plasmid is amplified in a PCR reaction, method 2 relies on restriction enzyme cleavage of the gene and vector, followed by ligation. In both methods, together with the mutation, a restriction enzyme marker site is introduced into the target gene by silent mutations, to allow fast and convenient screening for the presence of the mutation (,,). Because random mutagenesis and directed evolution are being widely used for protein engineering (), a method for random mutagenesis by PCR is described, using spiked oligonucleotides, which contain a mixture of all four nucleotides, to a certain degree (). This technique allows randomization of a small part of a gene, at a level that can be chosen at will, and is a convenient alternative to cassette mutagenesis methods described previously (,). The protocol described here allows the construction of large mutant libraries (104-105 clones). Because wild-type (WT) alleles are efficiently excluded from transformants, screening for the presence of the mutational primer is not necessary.
In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated. To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required. We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours. Using a 96-well format, the whole process can be performed automatically by a pipetting robot. Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours. The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates. Typical yields are 3–8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface. From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.
This chapter contains sections titled: Introduction Biology of restriction/modification systems Biochemical properties of type II restriction endonucleases Applications for type II restriction endonucleases Setting the stage for protein engineering of type II restriction endonucleases Design of Restriction Endonucleases with New Specificities Rational design Evolutionary design of extended specificities
AbstractMethods for the efficient overexpression and purification of recombinant proteins are of paramount importance for biotechnology. In particular, for the era of functional genomics that we have entered after sequencing complete genomes, this has become a routine matter. High-throughput protein purification will, therefore, become a key technology to unravel the function of gene products (Fig. 1). To facilitate the procedure of protein purification, several tags to generate fusion proteins are available (e.g., polyHis, GST, MBP, CBP, and the like) for parallel purification using matrices coupled with affinity anchors, like Ni2+-nitrilotriacetic acid (Ni2+-NTA) which is a powerful chelating ligand for the purification of His6-tagged proteins under native conditions. Ni-NTA affinity matrices allow to purify the protein of interest contained in a crude protein mixture at a concentration of 1% in one step to more than 95% homogeneity (1). Schematic overview of the high-throughput protein purification method. Cells are grown, harvested, and lysed in 1-mL square well blocks. The His6-tagged variants are isolated by transferring the lysate to Ni-NTA coated microplates from which they are eluted after washing. A 1.5-mL cell culture typically yields 5-10 pmol recombinant protein. KeywordsElution BufferProtein PurificationNitrilotriacetic AcidRefrigerate CentrifugePurify Enzyme PreparationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
