Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis.
The two domains of yeast phosphoglycerate kinase were produced by recombinant techniques. The N-domain was obtained by the introduction of a termination codon at the position coding for Phe185, and the C-domain by a deletion in the gene of the coding sequence between Ser1 and Leu186. Both domains were efficiently expressed in yeast, the level for the C-domain being greater than that for the N-domain. Both domains were found to have a quasi-native structure; the C-domain retained its ability to bind nucleotides. Small local differences were detected in domain structure compared to that in the whole enzyme, probably due to the lack of interdomain stabilizing interactions. Nevertheless, such an approach provides direct evidence for independent folding of domains in a two-domain protein.
Minard
P
P
Laboratoire d'Enzymologie Physico-chimique et Moléculaire, Université de Paris-Sud, Orsay, France.
Hall
L
L
Betton
J M
JM
Missiakas
D
D
Yon
J M
JM
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005800:Genes, Fungal; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D010735:Phosphoglycerate Kinase