Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.
Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.
Calvin
M C
MC
INSERM U91, CNRS UA 607 Hôpital Henri Mondor, Créteil, France.
Blouquit
Y
Y
Garel
M C
MC
Prehu
M O
MO
Cohen-Solal
M
M
Rosa
J
J
Rosa
R
R
D000595:Amino Acid Sequence; D001731:Bisphosphoglycerate Mutase / Q000235:genetics*; D002845:Chromatography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008969:Molecular Sequence Data
D001731:Bisphosphoglycerate Mutase