Cloning, Characterization, and Heterologous Expression of the Saccharopolyspora Erythraea (Streptomyces Erythraeus) Gene Encoding an EF-hand Calcium-binding Protein

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1989 Oct 1

PMID 2676983

Citations 14

Authors

D G Swan

J Cortes

R S Hale

P F Leadlay

Affiliations

Soon will be listed here.

Abstract

The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.

Citing Articles

Streptomycetes: Attractive Hosts for Recombinant Protein Production.

Berini F, Marinelli F, Binda E Front Microbiol. 2020; 11:1958.

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Calcium Regulation of Bacterial Virulence.

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CabC, an EF-hand calcium-binding protein, is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in Streptomyces coelicolor.

Wang S, Fan K, Yang X, Lin Z, Xu X, Yang K J Bacteriol. 2008; 190(11):4061-8.

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Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin.

Reddy P, Prasad C, Reddy P, Reeder D, McKenney K, Jaffe H J Bacteriol. 2003; 185(17):5263-8.

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NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

Aitio H, Annila A, Heikkinen S, Thulin E, Drakenberg T, Kilpelainen I Protein Sci. 2000; 8(12):2580-8.

PMID: 10631973 PMC: 2144237. DOI: 10.1110/ps.8.12.2580.

References

Sundaralingam M, Bergstrom R, Strasburg G, Rao S, Roychowdhury P, Greaser M . Molecular structure of troponin C from chicken skeletal muscle at 3-angstrom resolution. Science. 1985; 227(4689):945-8. DOI: 10.1126/science.3969570. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Norris V, Seror S, Casaregola S, Holland I . A single calcium flux triggers chromosome replication, segregation and septation in bacteria: a model. J Theor Biol. 1988; 134(3):341-50. DOI: 10.1016/s0022-5193(88)80065-1. View

Davis T, Urdea M, Masiarz F, Thorner J . Isolation of the yeast calmodulin gene: calmodulin is an essential protein. Cell. 1986; 47(3):423-31. DOI: 10.1016/0092-8674(86)90599-4. View

Kretsinger R, Nockolds C . Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973; 248(9):3313-26. View

Flinta C, Persson B, Jornvall H, von Heijne G . Sequence determinants of cytosolic N-terminal protein processing. Eur J Biochem. 1986; 154(1):193-6. DOI: 10.1111/j.1432-1033.1986.tb09378.x. View

Deng Z, Kieser T, Hopwood D . Activity of a Streptomyces transcriptional terminator in Escherichia coli. Nucleic Acids Res. 1987; 15(6):2665-75. PMC: 340676. DOI: 10.1093/nar/15.6.2665. View

Szebenyi D, Moffat K . The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins. J Biol Chem. 1986; 261(19):8761-77. View

Means A, Tash J, Chafouleas J . Physiological implications of the presence, distribution, and regulation of calmodulin in eukaryotic cells. Physiol Rev. 1982; 62(1):1-39. DOI: 10.1152/physrev.1982.62.1.1. View

10.

Tabor S, Richardson C . A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985; 82(4):1074-8. PMC: 397196. DOI: 10.1073/pnas.82.4.1074. View

11.

Hewick R, Hunkapiller M, HOOD L, Dreyer W . A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981; 256(15):7990-7. View

12.

Teintze M, Inouye M, Inouye S . Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis. J Biol Chem. 1988; 263(3):1199-203. View

13.

Iwasa Y, Yonemitsu K, Matsui K, Fukunaga K, Miyamoto E . Calmodulin-like activity in the soluble fraction of Escherichia coli. Biochem Biophys Res Commun. 1981; 98(3):656-60. DOI: 10.1016/0006-291x(81)91164-5. View

14.

Kretsinger R . Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem. 1980; 8(2):119-74. DOI: 10.3109/10409238009105467. View

15.

Suggs S, Wallace R, Hirose T, Kawashima E, Itakura K . Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin. Proc Natl Acad Sci U S A. 1981; 78(11):6613-7. PMC: 349099. DOI: 10.1073/pnas.78.11.6613. View

16.

Tsao S, Brunk C, Pearlman R . Hybridization of nucleic acids directly in agarose gels. Anal Biochem. 1983; 131(2):365-72. DOI: 10.1016/0003-2697(83)90185-9. View

17.

Staden R . Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982; 10(15):4731-51. PMC: 321125. DOI: 10.1093/nar/10.15.4731. View

18.

Cohen S, Chang A, Hsu L . Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972; 69(8):2110-4. PMC: 426879. DOI: 10.1073/pnas.69.8.2110. View

19.

Kretsinger R, Rudnick S, Weissman L . Crystal structure of calmodulin. J Inorg Biochem. 1986; 28(2-3):289-302. DOI: 10.1016/0162-0134(86)80093-9. View

20.

Matthes H, Zenke W, Grundstrom T, Staub A, Wintzerith M, Chambon P . Simultaneous rapid chemical synthesis of over one hundred oligonucleotides on a microscale. EMBO J. 1984; 3(4):801-5. PMC: 557430. DOI: 10.1002/j.1460-2075.1984.tb01888.x. View