Molecular cloning and primary structure analysis of porcine pancreatic alpha-amylase

A cDNA library was constructed in a Uni-ZAP XR vector using mRNA isolated f rom porcine pancreas. A full-length alpha-amylase cDNA was obtained using a combination of library screening and nested polymerase chain reaction. Seq uencing of the clone revealed a 1536-nucleotide (nt) open reading frame enc oding a protein of 496 amino acid (aa) residues with a signal peptide of 15 aa. The calculated molecular mass of the enzyme was 55 354 Da, in accordan ce with those of the purified porcine pancreatic cc-amylase forms (PPAI and PPAII) as determined by mass spectrometry. A comparison of the deduced aa sequence with published peptidic sequences of PPAI identified a number of m ismatches. The sequence of the cDNA reported here provides a sequence refer ence for PPA in excellent agreement with the refined three-dimensional stru ctures of both PPAI and PPAII. No evidence for a second variant was found i n the cDNA library and it is most likely that PPAI and PPAII are two forms of the same protein. The primary structure of PPA shows high homology with human, mouse and rat pancreatic alpha-amyiases. The 304-310 region, corresp onding to a mobile loop involved in substrate binding and processing near t he active site, is fully conserved. (C) 1999 Published by Elsevier Science B.V. All rights reserved.