Expression of bovine intestinal calcium binding protein from a synthetic gene in Escherichia coli and characterization of the product

Intestinal calcium binding proteins (ICaBP's) constitute a group of small vitamin D inducible proteins considered to play an important role in the absorption of dietary calcium. The mammalian ICaBP's are representatives of the "EF-hand" family of calcium binding proteins. As a fi...

Full description

Saved in:
Bibliographic Details
Published in:Biochemistry (Easton) 1986-09, Vol.25 (19), p.5371-5377
Main Authors: BRODIN, P, GRUNDSTROM, T, HOFMANN, T, DRAKENBERG, T, THULIN, E, FORSEN, S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Biological and medical sciences
Biotechnology
Cattle
DNA Restriction Enzymes
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Genes
Genes, Synthetic
Genetic engineering
Genetic technics
Intestinal Mucosa - metabolism
Methods. Procedures. Technologies
Plasmids
S100 Calcium Binding Protein G - genetics
Transcription, Genetic
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Intestinal calcium binding proteins (ICaBP's) constitute a group of small vitamin D inducible proteins considered to play an important role in the absorption of dietary calcium. The mammalian ICaBP's are representatives of the "EF-hand" family of calcium binding proteins. As a first step in the application of protein engineering techniques to the study of structure-function relationships in mammalian ICaBP's, we have synthesized a gene encoding the minor A form (the native form lacking the two N-terminal amino acids) of bovine ICaBP employing a rapid, microscale gene synthesis technique based on "shotgun ligation" of sets of oligonucleotides. Expression of the synthetic gene from a plasmid containing the tac promoter in a lon protease deficient strain of Escherichia coli yielded the desired product at a level of about 1-2 wt % of total protein. During the purification of the ICaBP expressed in E. coli, a contaminant was strongly adhering to it but was efficiently removed by gel filtration after denaturation with urea. The minor A form of ICaBP produced in E. coli was characterized by its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by its total amino acid composition, partial amino acid sequence, UV absorption spectrum, and 360-MHz 1H NMR spectrum, showing beyond reasonable doubt its identity with the minor A form of ICaBP obtained from bovine intestines.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00367a004