title: High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin.
abstract: Caltractin is a member of the calmodulin superfamily of Ca(2+)-binding proteins that was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii. Human and mouse homologs to algal caltractin have been recently characterized. In the studies reported here, recombinant Chlamydomonas caltractin was expressed at high levels in Escherichia coli and purified to homogeneity. The use of the ompT-host BL21 proved critical for obtaining high yields of homogeneous full-length protein. Growth and purification protocols were optimized to allow reproducible and efficient production of tens of milligrams of pure protein from 1-liter cultures. Caltractin has a distinct UV spectrum which is largely dominated by the fine structure due to the 9 Phe residues. Unlike other members of the same protein family, the UV and the CD spectra do not change upon addition of Ca2+ to the apoprotein. However, the 1H NMR spectrum shows distinct changes upon Ca2+ binding, which are indicative of structural and/or dynamic changes largely reminiscent of other members of the calmodulin superfamily. Ca2+ binding measurements demonstrated the binding of four Ca2+ ions to caltractin with two higher affinity (Kd = 1.2 x 10(-6) M) and two lower affinity (Kd = 1.6 x 10(-4) M) sites. Caltractin is highly stable in both the apo- and the Ca(2+)-loaded states. The unusual stability of apocaltractin makes this protein highly suited for structural studies by multidimensional NMR aimed at understanding the structural and dynamic consequences of Ca2+ binding, and the molecular basis of Ca2+ signal transduction.
authors.affiliation: Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
authors.forename: C
authors.initials: C
authors.lastname: Weber
authors.forename: V D
authors.initials: VD
authors.lastname: Lee
authors.forename: W J
authors.initials: WJ
authors.lastname: Chazin
authors.forename: B
authors.initials: B
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D011994:Recombinant Proteins; C056608:caltractin; D002118:Calcium