Process for producing proteins using gram negative host cells

Details Process for producing proteins using gram negative host cells Process for producing proteins using gram negative host cells

1988-02-29

1993-12-07

Ellis, Joan

Chemistry: molecular biology and microbiology

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

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

4351723, 4352351, 4353201, 4352523, 43525238, 536 234, 536 232, 536 237, 530350, 935 10, 935 19, 935 29, 935 48, 935 56, 935 61, 935 43, C12P 2102, C12P 1934, C12N 1500, C12N 700, C12N 121, C07H 1512, C07K 300

Patent

active

052682706

DESCRIPTION:

BRIEF SUMMARY
DESCRIPTION

The invention concerns a process for the production of proteins using gram-negative host cells, into which a vector is introduced which contains at least one gene coding the desired protein, transcription of this gene and translation.
Gene-technological processes for the production of proteins have long been known. Preferably, microorganisms are used for this purpose which are easy to culture and which permit the extraction of the protein produced in a simple manner. A widely-used organism is E. coli which can very easily be cultured and the properties of which are very well known. It a disadvantage of this micro-organism species that, as a gram-negative micro-organism, they frequently does not discharge the proteins or polypeptides produced into the surroundings but, at most, gives them off into the periplasma. The working up is, therefore, laborious and can only take place with destruction of the microorganisms.
Admittedly, a plurality of proteins are secreted by gram-negative bacteria. It has been found, however, that, for the secretion, specific bacterial proteins are essential for the second translocation step through the structural genes membrane. The cloning of the corresponding structural genes led to the accumulation of these proteins in the periplasma of E. coli. The same effect is also described for the Haemophilus influenzas IgA-protease which is secreted extracellularly in the natural host but which, when it is cloned into E. coli, is found in the periplasm. Still more complex is the secretion of the E. coli haemolysine. Here, a total of 4 different protein components, which are coded on a single operon, are necessary.
It was, therefore, the object of the present invention to improve a process for the gene-technological production of proteins using gram-negative host cells so that the desired protein, after its formation in the host cell, is discharged from the cell and can then be obtained extracellularly from the culture medium.
This task was solved by a process for the gene-technological production of proteins with the use of gram-negative host cells in which a vector is introduced, which contains at least one gene coding the desired protein, transcription of this gene and translation, which is characterised in that for the extracellular obtaining of the protein, one inserts its coding gene into a vector which contains the IgA-protease precursor gene from micro-organisms of the species Neisseria, in such manner that the coding gene is positioned within the sequence of the IgA-protease precursor gene.
Various pathogenic types of bacteria of the species Neisseria, such as Neisseria gonorrheae and Neisseria meningitidis, which grow on the human raucous membrane, emit extracellular proteases, which are specific for hurian IgA 1. This immunoglobulin is, besides IgA 2, the main component of the secretory immunity which is to protect against infections by such pathogens. Related types of bacteria which are not pathogenic produce no IgA-proteases.
The IgA-protease gene obtained from Neisseria displays surprising properties. The IgA-protease formed or its precursor can be actively secreted not only in the natural host but also in foreign gramnegative host cells, such as Enterobacteriaceae. A single gene which codes a large precursor suffices for the production and extracellular secretion of IgA-protease even in foreign host cells. This precursor, which, with a molecular weight of 170 kd, is approximately 63 kd larger than the final extracellular IgA-protease, which only has a molecular weight-of 106 kd.
The precursor protein is converted into the final extracellular IgA protease during transport through the membrane of the gram negative host cell. Processing leading to the final product includes autoproteolytic activity of the enzyme. This autoproteolytic activity involves splitting off of some parts of the precursor molecule leading to the final, active form of the IgA protease. This precursor protein is coded by a DNA fragment with 5 Kb.
The precursor has 3 functional domains, a homog

REFERENCES:
patent: 4338397 (1982-07-01), Gilbert et al.
Kooeny et al. Proc. Natl. Acad. Sci. USA vol. 79 pp. 7881-7885 (1985).
Zemel-Dreasen Gine vol. 27 pp. 315-322 (1984).
Halter et al. EMBO vol. 3 pp. 1595-1601 (1984).
Pohlner et al. Nature vol. 335 pp. 458-462 (1987).

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