Secretion of outer membrane proteins of gram-negative bacteria b

Details Secretion of outer membrane proteins of gram-negative bacteria b Secretion of outer membrane proteins of gram-negative bacteria b

1996-07-18

1999-01-12

Jacobson, Dian C.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

4353201, 4352523, 435 698, 530350, 530825, 536 234, 4241921, C07K 14195, C07K 14245, C12N 1531, C12N 1562

Patent

active

058587276

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The invention relates to a process for producing outer membrane proteins recoverable from the culture supernatant of Gram-negative bacteria from Gram-positive host cells, a gene structure capable of expressing the outer membrane proteins, a vector containing a gene structure capable of expressing the outer membrane protein, Gram-positive host cells containing said gene structure, outer membrane proteins expressed by the gene structure and a method of using the outer membrane proteins.


BACKGROUND OF THE INVENTION

Proteins from the outer membrane of Gram-negative bacteria are primarily exposed on the cell surface of these bacteria and in the case of pathogenic bacteria serve as possible attack points for the recognition of the bacteria by the immune system of the infected host.
It is thus desirable, with the aid of purified outer membrane proteins by inoculation to achieve protection of the host against the corresponding bacterium (Smyth, C. J. (1985) Immunology of outer membrane proteins of Gram-negative bacteria. In: Immunology of the Bacterial Cell Envelope (Stewart-Tull, D. E. S., Davies, M.; eds.) John Wiley and Sons Ltd. Pp. 177-201). A further possible use of isolated membrane proteins is their use as antigen in diagnostic test systems.
One way for recovery of the proteins is their isolation from the outer membrane Gram-negative bacteria. It is, however, problematic in that case that the outer membrane proteins have a high affinity to the lipopolysaccharide localized in the outer membrane so that the proteins can only be separated with difficulty from these endotoxins (Nikaido, H., Vaara M. (1985) Molecular basis of bacterial outer membrane permeability. Microbio). Rev. 49, 1-32). It is desirable to provide a way of producing outer membrane proteins which utilizes Gram-positive bacteria for their synthesis and which have an outer membrane with no lipopolysaccharide. One can expect that with these organisms endotoxin free outer membrane proteins can be recovered.
There are already different outer membrane proteins of Gram-negative bacteria (e.g. OmpA, OmpF) which are expressed intracellularly in the Gram-positive bacterium Bacillus subtilis (Puohiniemi, R., Butcher, S., Tarkka, E., Sarvas, M., (1991) High level production of Escherichia coli outer membrane proteins OmpA and OmpF intracellularly in Bacillus subtilis. FEMS Micro-biol. Lett. 83, 29-34). It has also been sought to export outer membrane proteins through the cytoplasmic membrane of Gram-positive bacteria for which secretory proteins are used for channeling in the exportation step and are necessary for the subsequent membrane transport and are fused to the outer membrane proteins: for example, a fusion of the membrane protein with the 298 aminoacid secretory protein .alpha.-amylase Bacillus amyloliquefacines is effected. In all of these cases, the expected secretion or transport of such fused membrane proteins is not found to occur (Kallio, P., Simonen, M., Pavla, I., Sarvas, M. (1986), Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis, J. Gen. Microbiol. 132, 677-678; Puohiniemi, R., Simonen, M., Muttilainen, S., Himanen, J.-P., Sarvas, M. (1992), Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step., Mol. Microbiol. 6, 981-990; Simonen, M., Tarkka, E., Puohiniemi, R., Sarvas, M. (1992), Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides, FEMS (Microbiol. Lett. 100, 233-242). By contrast, one finds instead, after expression, an outer membrane protein of Escherichia coli together with the authentic Gram-negative signal peptide in Bacillus subtilis, a transport through its cytoplasmic membrane but with the protein remaining cell associated, i.e. not secreted into the culture medium (Meens, J., Frings, E., Klose, M., Freudi, R. (1993), An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cyto

REFERENCES:
Meens, J. et al. Applied & Environmental Microbiology 63(7): 2814-2820 (1997).
Meens et al. Molecular Microbiology 9(4) : 847-855 (1993).
Puohiniemi, R. et al. FEMS 106: 105-110 (1993).

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