Cell-free protein synthesis can now be routinely used for the rapid screening of protein expression at the microliter level using PCR-amplified templates. However, identification of the optimal expression construct for a target protein can still be a problem. A rapid cell-free procedure is described here for the systematic assessment of a range of diverse fusion tags on the expression and solubility of any given target protein. Overlap/extension PCR is used to fuse a library of T7 promoter (T7p)-tag fragments with a gene-T7terminator (T7ter) fragment to produce cell-free expression templates encoding different fusion proteins. These constructs are then expressed in a series of small-scale (50 μL) Escherichia... More
Cell-free protein synthesis can now be routinely used for the rapid screening of protein expression at the microliter level using PCR-amplified templates. However, identification of the optimal expression construct for a target protein can still be a problem. A rapid cell-free procedure is described here for the systematic assessment of a range of diverse fusion tags on the expression and solubility of any given target protein. Overlap/extension PCR is used to fuse a library of T7 promoter (T7p)-tag fragments with a gene-T7terminator (T7ter) fragment to produce cell-free expression templates encoding different fusion proteins. These constructs are then expressed in a series of small-scale (50 μL) Escherichia coli cell-free reactions and SDS-PAGE analysis is used to identify the optimal fusion tag(s). This screen is particularly useful for the identification of expression constructs for proteins that are normally poorly expressed or are insoluble.
