Abstract
A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.
Highlights
Biochemistry, and $MolecularBwlogy, Genentech, Inc., South Sun Francisco, California94080 synthesizer (Biosearch) by the solid phase phosphite method (Beaucage and Caruthers, 1981; Matteucci and Caruthers, 1981; Froehler and Matteucci, 1983)
(BPTI)was fused to the coding sequence for thEe sch- Cloning and Mutagenesis-A 38-mer based on the first 38 nucleoerichia coli alkaline phosphatase signal peptideand tides of the Bovine pancreatic trypsin inhibitor (BPTI) coding sequence (Anderson and Kingston, 1983)
The BPTI produced by E. coli had three disulfide bonds that appeared to be aL, 1985)
Summary
In order to produce mature BPTI in the periplasm of E. coli, the coding region for BPTI was precisely fused to theE. coli alkaline phosphatase promoterand signal sequencecoding region (Figs. 1 and 2). Cys-14-Cys-38 or the Cys-30-Cys-51 disulfide bonds have been reduced can be made in uitro. These alternative states of BPTI exhibit full activity toward trypsin (States et al, 1980; Kosen et al, 1981; States et al, 1984; Creighton and extracts that irreversibly inhibited trypsin (Fig. 3). It was important, to examine activity was present in extracts from pBR322-transformed the BPTIproduced by E. coli to see if all the disulfide bonds cells grown under the same conditions
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