Construction and expression of synthetic wild-type and mutant genes encoding porcine pancreatic colipase: tryptophan fluorescence studies.
Based on the known (95-residue) amino acid (aa) sequence of porcine pancreatic colipase (CLP), a cofactor of pancreatic lipase, a 297 bp gene was designed and assembled from eight synthetic, overlapping DNA fragments. Optimized for expression in bacteria, the CLP-encoding gene (CLP) was inserted into the lacZ gene fragment contained in the small expression vector, pUC8, and cloned in Escherichia coli JM109. Expression of this construct yielded a protein approx. 11 kDa in size, equivalent to CLP, with an Mr of 10,336, plus ten additional amino acids at the N-terminus. The recombinant CLP (reCLP) was solubilized from bacterial inclusion bodies and then purified and refolded. A mutant CLP gene, changing Tyr-55 to Trp, was then constructed by site-directed mutagenesis. Since porcine CLP contains no Trp, this strategy provided a protein with an internal fluorescent probe for biophysical studies. The presence of Trp in the mutant protein was confirmed using fluorescence spectroscopy. Both wild-type (wt) and mutant reCLP reacted on Western blots with an affinity-purified rabbit anti-CLP antibody, raised against native CLP. The Tyr-55 to Trp exchange did not affect the activity of reCLP. Fluorescence studies of the interaction between reCLP and the bile salt, taurodeoxycholate (TDOC), showed that Trp-55 in the hydrophobic binding site of mutant reCLP inserted into the interior of the bile salt micelle.
Ernst
E G
EG
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, OH.
Behnke
W D
WD
D000595:Amino Acid Sequence; D000818:Animals; D001433:Bacteriolysis; D001483:Base Sequence; D003001:Cloning, Molecular; D003089:Colipases / Q000235:genetics* / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013552:Swine; D014364:Tryptophan / Q000378:metabolism*
D003089:Colipases; D014364:Tryptophan; D004247:DNA