Structure—Function analyses of mammalian cellular retinol-binding proteins by expression in Escherichia coli

This chapter discusses the structural and functional analyses of mammalian cellular retinol-binding proteins by expression in Escherichia coli. Two desirable components of prokaryotic expression vectors are (1) inducible promoters that can direct efficient transcription of foreign cDNAs and (2) translational control elements that allow efficient initiation of translation of the foreign mRNA transcript to occur. The bacterial strain used for expression of the recombinant protein must be selected with several caveats in mind. First, the induction system used must be compatible with the bacterial phenotype. Second, E. coli proteases such as the product of the lon gene may cause proteolysis of some foreign proteins. Expression of these proteins may be improved by using strains that are protease deficient. Third, variables that can affect the efficiency of production of recombinant proteins include incubation temperature, growth medium, cell density, timing of induction, and the length of fermentation after induction of foreign protein synthesis. Finally, it should be noted that E. coli cannot support many critical posttranslational modifications of mammalian proteins.