Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin.
A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.
Reddy
Prasad T
PT
Bioprocess Engineering Group, Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. prasad.reddy@nist.gov
Prasad
C Rama
CR
Reddy
P Hemalatha
PH
Reeder
Dennis
D
McKenney
Keith
K
Jaffe
Howard
H
Dimitrova
Mariana N
MN
Ginsburg
Ann
A
Peterkofsky
Alan
A
Murthy
P Suryanarayana
PS
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D005057:Eukaryotic Cells; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D002147:Calmodulin; D002118:Calcium