Codon-Optimized Cloning, Expression and Characterization of the C-Terminal Region of Human Apoptotic Protein GADD34 in Escherichia coli

Abstract

The human GADD34 (Growth Arrest and DNA Damage-inducible 34) is the product of an apoptosis- and DNA-damage-inducible gene. The C-terminus domain of GADD34 is highly homologous to HSV-1 g1 34.5, HSV-2 and the African swine fever virus virulence-associated factor NL-S. Among these viral proteins, HSV-1 g 34.5 protein is known to prevent apoptosis of viral-infected cells. Because of the difficulty in expressing GADD34 protein or any of its fragments, including the C-terminus (amino acids 533-632) in E. coli, partially due to sub-optimal expression of eukaryotic codons in prokaryotic E. coli, we used a codon-optimized cloning scheme to construct the eukaryotic gene that codes for GADD34533-632. We derived a novel PCR protocol to assemble 20 oligonucleotides into the synthetic GADD34533-632 gene. The clear advantage of using this protocol is that the assembled gene is without the mutation and deletion that are usually of major problem in constructing synthetic genes. The synthetic GADD34533-632 gene was cloned, expressed, and purified in large quantity. We obtained approximately 50 mg of GADD34533-632 protein per liter minimum-medium culture. To our knowledge, this is the first report of a large-scale production of the C-terminus of GADD34. The production and purification of GADD34533-632 in large quantity are essential for structure determination as well as for understanding its interactions with other proteins such as phosphatase 1-a using NMR spectroscopy and other biophysical methods.