Expression in Echerichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein

M. D. Gross, Rajiv Kumar, W. Hunziker

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18 Citations (Scopus)

Abstract

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.

Original languageEnglish (US)
Pages (from-to)14426-14432
Number of pages7
JournalJournal of Biological Chemistry
Volume263
Issue number28
StatePublished - 1988

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Calbindins
Rats
Brain
Calcium
Calcium-Binding Proteins
Recombinant Proteins
Proteins
Isoelectric Point
EF Hand Motifs
Amino Acids
Protein Sequence Analysis
Isoelectric Focusing
Molecular mass
Electrophoresis
Chemical analysis
Vitamin D
Chickens
Assays
Complementary DNA
Molecular Weight

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression in Echerichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein. / Gross, M. D.; Kumar, Rajiv; Hunziker, W.

In: Journal of Biological Chemistry, Vol. 263, No. 28, 1988, p. 14426-14432.

Research output: Contribution to journalArticle

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