Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.
Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.
Ray
S
S
Department of Cell Biology, Stanford University School of Medicine, CA 94305.
Zozulya
S
S
Niemi
G A
GA
Flaherty
K M
KM
Brolley
D
D
Dizhoor
A M
AM
McKay
D B
DB
Hurley
J
J
Stryer
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D003461:Crystallography; D004247:DNA / Q000235:genetics; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry; D051603:Recoverin; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014961:X-Ray Diffraction
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D051603:Recoverin; D051597:Hippocalcin; D014364:Tryptophan; D004247:DNA; D002118:Calcium