29] Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli

Abstract

Publisher Summary The chapter describes a general method for producing eukaryotic proteins in Escherichia coli using the fusion protein expression vector pLcII(nic – ) and subsequent purification and cleavage methods using blood coagulation factor X a . The foreign coding sequence can be joined in phase to a short coding sequence of a highly expressed E. coli gene so that a hybrid protein is produced. The short segment of E. coli gene directs the folding of the mRNA in the vicinity of the ribosome-binding site, and thereby ensures high translational efficiency. If the fusion protein, thus, produced can be cleaved specifically at the junction of the two sequences by an appropriate enzymatic or chemical method, the authentic protein will then be liberated. A fusion protein expression vector pLclI(nic-) is constructed. This plasmid has multiple cloning sites downstream of a short coding sequence from the λ cII gene, which is under the control of the strong leftward (PL) promoter of λ phage. The following proteins have been produced in this way at the level of 5–10% of total E. coli cellular protein: human β-globin and human α-globin, human myoglobin. In a second stage, a sequence encoding the tetrapeptide Ile-Glu-Gly-Arg at the junction in the fusion proteins is inserted. The chapter discusses cloning into the pLclI(nic – ) fusion protein expression vector, protein purification, the isolation of factor X from bovine blood, and several other related concepts. The pLcII(nic – ) fusion protein expression vector is a straightforward and general method for production of eukaryotic proteins in E. coli.