A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli.
A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
Little
S
S
Department of Microbial Systems, Celltech Group plc, Slough, Berks, U.K.
Campbell
C J
CJ
Evans
I J
IJ
Hayward
E C
EC
Lilley
R J
RJ
Robinson
M K
MK
D000469:Alkaline Phosphatase / Q000235:genetics*; D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; C032443:prorennin; D012085:Chymosin