Mutagenesis of human fibrinogen gamma chain 259-411 synthesized in E. coli: further characterization of the role of the disulfide bond CYS326-CYS339 in calcium binding.
We have produced human fibrinogen gamma 259-411 in Escherichia coli in order to study the relationship between the calcium binding activity of the polypeptide and the integrity of the disulfide bond cysteine326-cysteine339. The polypeptide was produced from a plasmid expression vector at approximately 5 micrograms per milliliter of bacterial culture. The identity of the polypeptide was confirmed by N-terminal amino acid sequence analysis. The expression vector was modified by oligonucleotide directed mutagenesis to remove the nucleotides encoding cysteines gamma 326 and gamma 339. The calcium binding activity of wild-type and altered polypeptides were compared using a solid phase assay. Our results indicate that removal of the two cysteine residues had no appreciable effect on calcium binding activity. We conclude that the integrity of the disulfide bond cysteine326-cysteine339 is not critical for calcium binding to gamma 259-411.
Bolyard
M G
MG
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Lord
S T
ST
D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000378:metabolism
D004220:Disulfides; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D005340:Fibrinogen; D003545:Cysteine; D002118:Calcium