Process for producing extracellular proteins in bacteria

Details Process for producing extracellular proteins in bacteria Process for producing extracellular proteins in bacteria

1997-06-02

2001-01-09

Minnifield, Nita (Department: 1645)

Chemistry: molecular biology and microbiology

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

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

C435S069100, C435S320100, C435S220000, C435S824000, C536S023200

Reexamination Certificate

active

06171823

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK95/00498 filed Dec. 8, 1995, and claims priority under 35 U.S.C. 119 of Danish application 1411/94 filed Dec. 9, 1994, the contents of which applications are fully incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a method of producing an extracellular protein in a bacterium, as well as to a DNA construct and a recombinant vector comprising a DNA sequence encoding said protein.
BACKGROUND OF THE INVENTION
Prokaryotic organisms provided with both an inner and outer cell membrane such as gram-negative bacteria only rarely secrete proteins out of the cell into the surrounding medium. Such proteins which do not remain in the cytoplasm are usually exported across the cytoplasmic membrane into the periplasmic space but do not cross the outer cell membrane. However, more recently examples have been found of proteins which are truly secreted from gram-negative bacteria.
Thus,
Lysobacter enzymogenes
secretes an alkaline phosphatase (cf. S. Au et al.,
J. Bacteriol.
173(15), 1991, pp. 4551-4557) and an &agr;-lytic protease. Attempts to express the &agr;-lytic protease in
E. coli
have resulted in production of the enzyme within cells as well as in the extracellular medium (cf. J. L. Silen et al.,
J. Bacteriol.
171(3), 1989, pp. 1320-1325).
Likewise,
Achromobacter lyticus
produces an extracellular protease, the primary structure of which appears from S. Tsunasawa et al.,
J. Biol. Chem.
264(7), 1989, pp. 3832-3839. The gene encoding
A. lyticus
protease I was cloned in
E. coli
in which the enzyme was exported to the periplasm rather than secreted into the culture medium (cf. T. Ohara et al.,
J. Biol. Chem.
264(34), 1989, pp. 20625-20631).
SUMMARY OF THE INVENTION
It has now been found possible to obtain extracellular production of proteins from a heterologous host bacterium which does not readily translocate proteins out of the cells. Such extracellular production is accomplished by means of a prepropeptide or part of a prepropeptide of certain bacterial extracellular proteases.
Accordingly, the present invention relates to a method of producing an extracellular protein in a bacterium provided with an inner and outer cell membrane, the method comprising
(a) providing a recombinant vector including a DNA construct comprising a DNA sequence encoding the prepropeptide or part of the prepropeptide of a bacterial extracellular protease selected from the group consisting of
Achromobacter lyticus
protease I, Bacillus metalloproteases and Bacillus serine proteases preceding and operably connected to a DNA sequence encoding a desired protein,
(b) transforming cells of a bacterium provided with an inner and outer cell membrane with the recombinant vector of step (a),
(c) culturing the transformed cells of step (b) under conditions permitting expression of said DNA insert and leakage of the bacterial extracellular protease propeptide fused to the desired protein into the culture medium, and
(d) recovering the resulting protein from the medium.
In the present context, the term “bacterium provided with an inner and outer cell membrane” is intended to indicate a bacterium which has an inner, or cytoplasmic, membrane surrounding the cytoplasm of the cell as well as an outer membrane and a periplasmic space between the inner and outer membrane. In most such organisms, there are mechanisms (including signal sequences) permitting the translocation of expressed gene products across the inner membrane to the periplasm, while secretion of protein s through the outer membrane is far less common. The term “heterologous”, when applied to host cells, is intended to indicate that the host cell is one which does not, in nature, produce the protein in question.
The term “prepropeptide” is intended to indicate a peptide composed of a signal peptide (the prepeptide) and one or m ore peptide sequences present on a precursor form of the protein to be produced. If a part (or fragment) of a prepropeptide is employed in the method of the invention, it should be sufficient in length to have retained the ability of the full-length prepropeptide to bring about extracellular production of the protein of interest.
The term “bacterial extracellular protease” is intended to indicate a proteolytic enzyme produced in bacteria and secreted from bacterial cells. Examples of suitable bacterial extracellular proteases are
Achromobacter lyticus
protease I, Bacillus metalloproteases and Bacillus serine proteases, such as subtilisins.
The term “operably connected” is intended to indicate that the DNA sequence encoding the prepropeptide is transcribed together with the DNA sequence encoding the desired protein.
The protein produced by the present method may be either homologous or heterologous, either to the prepropeptide or to the host cell or both. Thus, it may be envisaged that the DNA sequence encoding the protein of interest may be preceded by a DNA sequence encoding a prepropeptide which, in nature, is connected to the protein-coding DNA sequence expressed in a host bacterium which does not naturally produce the protein. Alternatively, the DNA sequence encoding the protein of interest may be preceded by a DNA sequence encoding a prepropeptide which is not naturally connected to the protein-coding DNA sequence expressed in a host bacterium which produces the protein in nature. Furthermore, the DNA sequence encoding the protein of interest may be preceded by a DNA sequence encoding a prepropeptide which is not naturally connected to the protein-coding DNA sequence expressed in a host bacterium which does not naturally produce the protein.
The term “leakage” is intended to indicate that the protein produced by the present method is transported out of the cell either by secretion, i.e. translocation across both the inner and outer cell membrane, or by export of the protein to the periplasm followed by lysis of the outer membrane. The lysis of the outer membrane may be complete or partial, or the protein produced by the present method is transported out of the cell by export of the protein to the periplasm followed by release through the outer cell membrane.
In another aspect, the present invention relates to a method of producing an extracellular protein in a bacterium provided with an inner and outer cell membrane, in which method a bacterium provided with an inner and outer cell membrane and transformed with a recombinant vector including a DNA construct comprising a DNA sequence encoding the prepropeptide or part of the prepropeptide of a bacterial extracellular protease selected from the group consisting of
Achromobacter lyticus
protease I, Bacillus metalloproteases and Bacillus serine proteases preceding and operably connected to a DNA sequence encoding a desired protein, is cultured under conditions permitting expression of said DNA insert and leakage of the bacterial extracellular protease propeptide fused to the desired protein into the culture medium, and the resulting protein is recovered from the medium.
In a further aspect, the present invention relates to a recombinant expression vector including a DNA construct comprising a DNA sequence encoding the prepropeptide or part of the prepropeptide of a bacterial extracellular protease selected from the group consisting of
Achromobacter lyticus
protease I, Bacillus metalloproteases and Bacillus serine proteases preceding and operably connected to a DNA sequence encoding a desired protein. The vector is useful for transformation of a suitable host microorganism in the method of the invention described above.
In a still further aspect, the present invention relates to a DNA construct comprising a DNA sequence encoding the prepropeptide or part of the prepropeptide of a bacterial extracellular protease selected from the group consisting of
Achromobacter lyticus
protease I, Bacillus metalloproteases and Bacillus serine proteases preceding and operably connected to a DNA sequence encoding a desired protein. The DNA const

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