Application of high-throughput methodologies to the expression of recombinant proteins in E. coli

Despite the large body of knowledge accumulated on recombinant protein expression, production, primarily of eukaryotic proteins, remains a challenge. The biggest obstacle is in obtaining large amounts of a given protein in a correctly folded form. Several strategies are being used to increase both yields and solubility. These include expression with fusion proteins, co-expression with molecular chaperones or a protein partner, and use of multiple constructs for each protein. Any given method may help to increase expression and solubility for a given protein, but often more than one rescue strategy should be tried. To perform several different rescue strategies on multiple proteins, high throughout (HTP) methodologies are applied. This chapter presents HTP methodologies for DNA cloning in multiple expression vectors and expression screening to identify clones capable of producing soluble proteins.