Cloning and Expression of the Gene for a Novel Protein From Mycobacterium Smegmatis With Functional Similarity to Eukaryotic Calmodulin

Published

September 1, 2003

Author(s)

Prasad T. Reddy, C R. Prasad, P H. Reddy, D J. Reeder, K H. McKenney, H Jaffe, M N. Dimitrova, A Ginsburg, A Peterkofsky, P S. Murthy

Abstract

The calmodulin-like protein from Mycobacterium smegmatis (MSM-CAMLP) was purified to homogeneity with a yield of about 1 g from 70 g of cells. Peptide sequence analysis of the MSM-CAMLP was used to design degenerate oligonucleotides for the eight aminoterminal and eight internal amino acids. A PCR product derived from M. smegmatis genomic DNA and these oligonucleotides produced a 100 bp product, the sequence of which was used to synthesize a 30 bp probe for cloning the gene for MSM-CAMLP. DNA sequence analysis of such a clone coding for MSM-CAMLP revealed an open reading frame with 55 amino acids that matched all of the amino acids derived by Edman degradation. The gene for MSM-CAMLP was overexpressed and purified in E. coli as both a His-tagged protein and a fusion with GroEL in order to produce amounts suitable for biochemical studies. Nine amino acids (15%) of MSM-CAMLP are acidic. The MSM-CAMLP exhibited two important properties characteristic of the eukaryotic calmodulin: stimulation of eukaryotic phosphodiesterse in a calcium-dependent manner and reaction with anti-bovine brain calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of MSM-CAMLP. Ultracentrifuge studies excluded the possibility that calcium promotes oligomerization of MSM-CAMLP. Thus, M. smegmatis CAMLP appears to be functionally similar to the eukaryotic calmodulin although it has a different primary structural organization. We propose that this bacterial CAMLP is a progenitor of eukaryotic calmodulin.

Citation

Journal of Bacteriology

Volume

185

Issue

17

Pub Type

Journals

Keywords

calmodulin antagonist, calmodulin like protein, drug resistant mycobacteria, trifluoperazine

Citation

Reddy, P. , Prasad, C. , Reddy, P. , Reeder, D. , McKenney, K. , Jaffe, H. , Dimitrova, M. , Ginsburg, A. , Peterkofsky, A. and Murthy, P. (2003), Cloning and Expression of the Gene for a Novel Protein From Mycobacterium Smegmatis With Functional Similarity to Eukaryotic Calmodulin, Journal of Bacteriology (Accessed April 19, 2024)