Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.
The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.
Talarico
T L
TL
Department of Molecular Genetics and Microbiology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Ray
P H
PH
Dev
I K
IK
Merrill
B M
BM
Dallas
W S
WS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016477:Artifacts; D001483:Base Sequence; D003001:Cloning, Molecular; D017854:Diphosphotransferases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005492:Folic Acid / Q000096:biosynthesis; D015870:Gene Expression; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D005492:Folic Acid; D010770:Phosphotransferases; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase