Codon-optimized cloning, expression and characertization of the C-terminal region of human apoptotic protein GADD34 in Escherichia coli.
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 gamma-1 34.5, HSV-2 and the African swine fever virus virulence-associated factor NL-S. Among these viral proteins, HSV-1 gamma 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 GADD34(533-632). We derived a novel PCR protocol to assemble 20 oligonucleotides into the synthetic GADD34(533-632) gene. The clear advantage of using this protocol is that the assembled gene is without the mutation and deletion that are usually of a major problem in constructing synthetic genes. The synthetic GADD34(533-632) gene was cloned, expressed, and purified in large quantity. We obtained approximately 50 mg of GADD34(533-632) 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 GADD34(533-632) in large quantity are essential for structure determination as well as for understanding its interactions with other proteins such as phosphatase 1-alpha using NMR spectroscopy and other biophysical methods.
Yu
Ping
P
Protein-Nucleic Acid Interactions Section, Structural Biophysics Laboratory, NCI-Frederick, Frederick, Maryland 21702, USA.
Neamati
Nouri
N
Tarasov
Sergey
S
He
Qin
Q
Sheikh
M Saeed
MS
Wang
Yun-Xing
YX
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D000943:Antigens, Differentiation; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D054645:Protein Phosphatase 1; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000943:Antigens, Differentiation; D018797:Cell Cycle Proteins; D003062:Codon; D011506:Proteins; D011994:Recombinant Proteins; C087519:PPP1R15A protein, human; D054645:Protein Phosphatase 1