title: Expression of the protease inhibitor ecotin and its co-crystallization with trypsin.
abstract: We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0110.
authors.forename: M E
authors.initials: ME
authors.lastname: McGrath
authors.forename: T
authors.initials: T
authors.lastname: Erpel
authors.forename: M F
authors.initials: MF
authors.lastname: Browner
authors.forename: R J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D003461:Crystallography; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011480:Protease Inhibitors / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D014357:Trypsin / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014357:Trypsin