Expression, purification and activity measurements of pancreatic proteases

Abstract

The pancreas secretes four types of proteolytic digestive proenzymes, which after activation give rise to trypsin, chymotrypsin, elastase and carboxypeptidase. In humans, three trypsinogen isoforms, cationic trypsinogen (PRSS1), anionic trypsinogen (PRSS2) and mesotrypsinogen (PRSS3); four chymotrypsinogen isoforms, chymotrypsinogen B1 (CTRB1), chymotrypsinogen B2 (CTRB2), chymotrypsinogen C (CTRC), and chymotrypsinlike enzyme-1 precursor (CTRL1) and at least three proelastase isoforms, proelastase 2A (ELA2A), proelastase 3A (ELA3A) and proelastase 3B (ELA3B) are present in the pancreatic juice. Expression of the proelastase 1 (ELA1) gene is evolutionarily suppressed in humans by mutations in the promoter region. The product of the proelastase 2B (ELA2B) gene has not been identified in pancreatic tissue or juice yet and experiments with recombinantly expressed proelastase 2B (ELA2B) indicate that this isoform, if exists, may be catalytically inactive. There are three digestive procarboxypeptidase isoforms expressed in the human pancreas, procarboxypeptidase A1 (CPA1), procarboxypeptidase A2 (CPA2) and procarboxypeptidase B (CPB), also referred to as procarboxypeptidase B1 (CPB1). Here we describe methods we use in our laboratory for the expression, purification, activation and activity measurement of human digestive proteases. Although not tested in a systematic manner, the methodology should be generally applicable to most mammalian pancreatic digestive proteases.