Expression and purification of human calbindin D28k.
Calbindin D28k is a protein abundant in the mammalian central nervous system and in epithelial tissue involved in Ca2+ transport. Human calbindin D28k was cloned into a Pet3a vector and expressed in Escherichia coli. The protein was purified in three steps: (i) heat precipitation of bacterial proteins, (ii) ion-exchange chromatography on a DEAE-cellulose column in the presence of calcium, and (iii) ion-exchange chromatography on a DEAE-Sephacel column in the presence of EDTA. The protein was then supplemented with calcium and dialyzed against neutral water. The final yield was 20-50 mg of pure, homogeneous calcium-loaded calbindin D28k per liter of bacterial culture. The identity and purity of the protein were confirmed by immunoblotting, SDS-polyacrylamide gel electrophoresis, and agarose gel electrophoresis in the absence and presence of calcium and 1H NMR spectroscopy. The entire expression and purification protocol takes only 3 days and is easy to scale up and down. It was designed to minimize degradation and deamidation.
Thulin
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Physical Chemistry 2, Lund University, Lund, Sweden.
Linse
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D000595:Amino Acid Sequence; D001483:Base Sequence; D064092:Calbindin 1; D064026:Calbindins; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular / Q000379:methods; D016384:Consensus Sequence; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
C576498:CALB1 protein, human; D064092:Calbindin 1; D064026:Calbindins; D017931:DNA Primers; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G