An expression system for trypsin.
The eukaryotic serine protease, rat anionic trypsin, and various mutants created by site-directed mutagenesis have been heterologously expressed in Escherichia coli. The bacterial alkaline phosphatase (phoA) promoter was used to control the expression of the enzymes in an induced or constitutive fashion. The DNA coding for the eukaryotic signal peptide of pretrypsinogen was replaced with DNA coding for the phoA signal peptide. The phoA signal peptide successfully directs the secretion of the mammalian trypsinogen to the periplasmic space of E. coli. Active trypsin was expressed in the periplasm of E. coli by deleting the DNA coding for the activation hexapeptide of the zymogen. The activity of trypsin in the periplasm suggests that the enzyme is correctly activated and has folded such that the 12 cysteine residues involved in the six disulfide bonds of rat anionic trypsin have paired correctly. A transcription terminator increased the level of expression by a factor of two. However, increasing the copy number of the plasmid decreased the levels of expression. Localization of the active enzyme in the periplasm allows rapid screening of modified trypsin activities and facilitates the purification of protein to homogeneity and subsequently to crystallinity.
Vasquez
J R
JR
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Evnin
L B
LB
Higaki
J N
JN
Craik
C S
CS
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D014357:Trypsin / Q000235:genetics* / Q000302:isolation & purification; D014362:Trypsinogen / Q000235:genetics
D014362:Trypsinogen; D014357:Trypsin