Methods in Enzymology Vol.282 Vitamins and Coenzymes, Part L

This chapter describes the generation and characterization of cellular retinoic acid-binding proteins from Escherichia coli (E. Coli) expression systems. The cellular retinoic acid-binding proteins (CRABP)-II is expressed in the protease-deficient E. coli strain BL21(DE3) (lon- ompT-). This prevents proteolysis of the CRABP-II, which has been observed in other E. coli strains such as JM101. CRABP-I has been successfully expressed in E. coli strains BL21(DE3) and JM101. Significant expression can be obtained in a variety of media. The enhanced solubilization of CRABP expressed in a supplemented, M9-based medium. The expression and purification of CRABP can be evaluated by denaturing SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis on a 15% (w/v) polyacrylamide gel followed by Coomassie staining. Bacterial samples for SDS–PAGE analysis should be processed quickly because insults to the bacteria may artifactually induce the recA promoter. Both isoforms of CRABP exhibit an absorption peak at 280 nm typical of tryptophan-containing proteins. Analysis of purified CRABP by sequential Edman degradation can be a measure of protein purity, and should reproduce the expected sequence.

• Series: Methods in Enzymology (Book 282)
• Hardcover: 505 pages
• Publisher: Academic Press
• Language: English
• ISBN-10: 0121821838
• ISBN-13: 978-0121821838
• Product Dimensions: 6 x 1.2 x 9 inches

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