Protein fusions for the translocation of apo-protein into the pe

Details Protein fusions for the translocation of apo-protein into the pe Protein fusions for the translocation of apo-protein into the pe

1995-11-13

1999-10-05

Carlson, Karen Cochrane

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 691, 435325, 4353201, 536 231, C12Q 168, C12P 2106, C07H 1700

Patent

active

059622229

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

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.


BACKGROUND OF THE INVENTION

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.


SUMMARY OF THE INVENTION

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.
According to a first aspect of the present invention there is provided a method of protein synthesis which comprises provision of a genetic unit comprising a nucleotide sequence coding for a pre-form of an apo-protein, synthesizing said pre-form apo-protein in a cytoplasmic region of a cell, and translocating said synthesised pre-form apo-protein to the periplasmic region of said cell, so as to permit constitution in said periplasmic region of a signal-processed apo-protein which can then be converted to the corresponding holo-protein.
According to a second aspect of the present invention, there is provided a genetic precursor unit which comprises a nucleotide sequence coding for a pre-form apo-protein, said nucleotide sequence being such that said pre-form apo-protein is translocatable from a cytoplasmic region of a cell to the periplasmic region of said cell, so as to permit constitution in said periplasmic region of a processed apo-protein and conversion into a corresponding holo-protein.
The genetic unit is preferably suitable for expression in the cytoplasmic region of a bacterial host cell, such as an E.coli host cell. The galK-(.lambda.ch1D-pg1)(.lambda.Bam N+CI857 H1)!.
A preferred apo-protein comprises a cytoplasmic cytochrome, which typically comprises the soluble core domain of cytochrome b.sub.5 of liver endoplasmic reticulum.
Cytochrome b.sub.5 of the endoplasmic reticulum of mammalian liver is a well characterised, small haemoprotein of 16.7 kD which plays a central role in a variety of electron transfer reactions related to fatty acid desaturation, redox cycling of oestrogen and reduction of cytochrome P-450 reductase.
The hepatic cytochrome b.sub.5 is composed of two domains, the above-described soluble, enzymatically active, haem-containing globular core (b.sub.5) of about 12 kD and a smaller carboxy terminal tail anchored in the microsomal membrane. Within the tail portion, a stretch of 23 hydrophobic amino acid residues, also definable as an "insertion" sequence, autonomously and post-translationally integrates cytochrome b.sub.5 into the lipid bilayer of the endoplasmic reticulum such that the active core domain is laterally disposed from the reticulum facing the cytoplasm.
It is preferred that the genetic unit further comprises a nucleotide sequence which codes for an amino-terminal signal peptide (the pre region of pre-form apo-protein) which is recognised by the cell to direct the pre-form apo-protein to the cytoplasmic membrane and thence subsequently translocated into the periplasm of the cell. The signal peptide typically comprises E.coli alkaline phosphatase, which is advantageously in linkage with cytochrome b.sub.5 (apo-protein). The method therefore preferably furt

REFERENCES:
Gallagher et al. 1992 Appl Microbiol Biotechnol 38:77-83.
Page et al 1990 Mol. Microbiol. 4(7) 1181-1192.
Stader et al 1990 Methods in Enzymology 185: 166-186.
M. Takahara et al., "Secretion of Human Superoxide Dismutase in Escherichia coli", Biotechnology, vol. 6, No. 2, pp. 195-198, (1988).
A. Karim et al., "Efficient Bacterial Export of a Eukaryotic Cytoplasmic Cytochrome", Bio/Technology, vol. 11, No. 5, pp. 612-618, (1993).
V. Harding et al., "Processing of chimeric mammalian cytochrome b.sub.5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocation processing", Biochemical Journal, vol. 293, No. 3, pp. 751-756, (1993).

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