Expression, purification, and spectroscopic studies of the guanine nucleotide exchange domain of human elongation factor, EF-1beta.
Two guanine nucleotide exchange domains, corresponding to the C-terminal region of the human translational elongation factor EF-1beta (which consists of 225 amino acids), were produced by DNA recombinant overexpression techniques in Escherichia coli. We describe here a fast and efficient method for purifying these two protein fragments and for concentrating their solutions rapidly to a level as high as 25 mg/ml. This technique permitted the isolation of 20-30 mg of pure, native protein per liter of bacterial culture. Both fragments were able to form a complex with their natural substrate, elongation factor EF-1alpha, as detected by gel filtration experiments. The domain of 110 residues was slightly more active than the 91-amino-acid domain in guanine nucleotide exchange assays. Folding and stability of the two C-terminal domains were explored by circular dichroism (CD) and NMR spectroscopy. In spite of optimal conditions concerning NaCl concentration, temperature, and pH, during the NMR experiments both proteins showed signs of aggregation after approximately 7 days at 303 degreesK, a time period and temperature required for future heteronuclear NMR experiments. Also, the longer fragment suffered from proteolysis in the N-terminal region, suggestive of flexibility in that part of the structure. The secondary structure content for these two EF-1beta fragments was estimated, using data from both CD and NMR. The results of both methods agree very well and indicate for each fragment the presence of approximately 20% alpha-helix and approximately 50% beta-sheet. Elucidation of the three-dimensional structure of the exchange domain of EF-1beta by NMR spectroscopy appears therefore feasible.
Pérez
J M
JM
Department of Molecular Cell Biology, Sylvius Laboratory, Leiden University Medical Centre, Wassenaarseweg 72, Leiden, 2333 AL, The Netherlands.
Kriek
J
J
Dijk
J
J
Canters
G W
GW
Möller
W
W
D000818:Animals; D001156:Artemia; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006150:Guanine Nucleotides / Q000378:metabolism*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
D006150:Guanine Nucleotides; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins