Protein fusions for the translocation of apo-protein into the periplasmic space

#The present invention is concerned with protein synthesis and in particular with the production of recombinant fusions between cytochrome b.sub.5 and foreign proteins which can be translocated out of the cell into the periplasmic space of, for example, Escherichia coli.

#The mechanism by which a cytoplasmically-synthesised (recombinant) protein is translocated into the periplasmic space of Escherichia coli is known to occur by the workings of the signal hypothesis (see review by Stardler, J. A. and Silhavy, T. J., 1990, Methods in Enzymology, 167-187). Many such proteins are initially synthesised as precursor forms carrying extensions at their amino termini known as signal sequences (peptides). The signal sequence has the information required for selective translocation of the passenger part of the protein molecule across the cytoplasmic membranes of the bacteria. The signal is cleaved off soon after the periplasmically-deposited protein has gained its native biological fold and function.

#Using this approach we have developed a bacterial system which allows production of chimeric or fusion forms of coloured proteins. These proteins are secreted into the periplasm where they are afforded greater protection against potential degradation.

#The present invention will now be further illustrated, by way of example only, by reference to the accompanying FIGS. 1 to 6, wherein:

#FIGS. 1,2 and 3 illustrate expression vectors pAA-cyt, pSEC-cyt/C and PSEC-cyt/N of a genetic unit according to the present invention;

#FIGS. 4-6 illustrate the genetic sequences of the vectors respectively illustrated in FIGS. 1-3 (SEQ ID NOS 1 to 3, respectively); and

#FIG. 1 illustrates plasmid PAA-cyt constructed for the periplasmic production of non-fusion, secreted holo-cytochrome b.sub.5.

#FIG. 2 illustrates the plasmid PSEC-cyt/C constructed for linkage at the 3' terminus (C-terminus) of the soluble cytochrome b.sub.5 core gene with a foreign gene sequence, according to the present invention.

#FIG. 3 illustrates the plasmid PSEC-cyt/N for linkage at 5' terminus, between the C-terminus of alkaline phosphatase signal and the N-terminus of cytochrome b.sub.5 of the cytochrome b.sub.5 core gene, according to the present invention.