Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-03-30
2003-07-22
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S252300, C435S252330, C435S320100, C536S023100, C536S023500
Reexamination Certificate
active
06596510
ABSTRACT:
This Application is a 371 of PCT/EP98/04723 filed on Jul. 27, 1998, which claims benefit of German Application 197 32 829.6 filed on Jul. 30, 1997.
The present invention relates to processes for producing carrier-bound proteins, in particular S-layer proteins and modified S-layer proteins in pro- or eukaryotic host cells.
Crystalline bacterial cell surface layers (S-layers) form in many eubacteria and in most archaebacteria of the outermost cell wall component (Sleytr et al. (1988), Crystalline Bacterial Cell Surface Layers, Springer Verlag Berlin; Messner and Sleytr. Adv. Mikrob. Physiol. 33 (1992), 213-275). Most of the S-layer proteins known at present are composed of identical proteins or glycoproteins which have apparent molecular weights in the range from 40,000 to 220,000. The components of S-layers are self-assembling and most lattices have oblique (p2), square (p4) or hexagonal (p6) symmetry. The functions of bacterial S-layers are still not completely known but, on the basis of their localization on the cell surface, it is likely that the porous crystalline S-layers act mainly as protective coverings, molecular sieves or for promoting cell adhesion and surface recognition.
Genetic data and sequence information are known for various S-layer genes from microorganisms. A review is to be found in Peyret et al., Mol. Microbiol. 9 (1993), 97-109. Express reference is made to these data. The sequence of the gene sbsA coding for the S-layer protein of B.stearothermophilus PV72 and a method for cloning it are indicated by Kuen et al. (Gene 145 (1994), 115-20). B.stearothermophilus PV72 is a Gram-positive bacterium which is covered by a hexagonally arranged S-layer. The main component of the S-layer is a 128 kd protein which is the commonest protein in the cell, comprising approximately 15% of the total protein constituents. Various strains of B.stearothermophilus have been characterized and differ in the type of, S-layer lattice, the molecular weight and the glycosylation of the S-layer components (Messner and Sleytr (1992), supra).
German Patent Application DE-A 44 25 527 discloses the signal peptide-encoding section of the S-layer gene of B.stearothermophilus and the amino acid sequence derived therefrom. The cleavage site between the signal peptide and the mature protein is located between position 30 and 31 of the amino acid sequence. The signal peptide-encoding nucleic acid can be operatively linked to a protein-encoding nucleic acid and used for the recombinant production of proteins in a process in which a transformed host cell is prepared, the host cell is cultivated under conditions which lead to expression of the nucleic acid and to production and secretion of the polypeptide encoded thereby, and the resulting polypeptide is isolated from the culture medium. The host cells mentioned as preferred are prokaryotic organisms, in particular Gram-positive organisms of the genus Bacillus.
The international Patent Application PCT/EP97/00432 proposes the recombinant production of S-layer proteins and modified S-layer proteins in the cytoplasm of Gram-negative host cells.
It has now been found, surprisingly, not only that the recombinant production of S-layer proteins is possible in the cytoplasm of Gram-negative prokaryotic host cells, but also that recombinant expression comprising integration in the outer or the cytoplasmic membrane, secretion into the periplasm or/and secretion into the extracellular space can be carried out. It has additionally been found that recombinant expression of S-layer proteins also takes place in the eukaryotic host cells.
A first aspect of the present invention is thus a process for producing S-layer proteins, which comprises
(a) preparing a Gram-negative prokaryotic host cell which is transformed with a nucleic acid which codes for an S-layer protein and is operatively linked to a signal sequence which codes for a peptide which brings about integration of the S-layer protein in the outer membrane of the host cell, integration of the S-layer protein in the cytoplasmic membrane of the host cell, secretion of the S-layer protein into the periplasmic space of the host cell or/and secretion into the medium surrounding the host cell,
(b) cultivating the host cell under conditions leading to expression of the nucleic acid and to production of the polypeptide encoded thereby, and
(c) where appropriate isolating the resulting polypeptide from the outer membrane of the host cell, from the cytoplasmic membrane of the host cell from the periplasmic space of the host cell or/and from the medium surrounding the host cell.
A second aspect of the present invention is a process for producing S-layer proteins, which comprises
(a) preparing a eukaryotic host cell which is transformed with a nucleic acid which codes for an S-layer protein and is preferably operatively linked to a signal sequence which brings about integration of the S-layer protein in the cytoplasmic membrane of the host cell, integration of the S-layer protein into an organelle of the host cell or/and secretion into the medium surrounding the host cell,
(b) cultivating the host cell under conditions leading to expression of the nucleic acid and to production of the polypeptide encoded thereby, and
(c) where appropriate isolating the resulting polypeptide from the cytoplasmic membrane of the host cell, from an organelle of the host cell or/and from the medium surrounding the host cell.
It has been found, surprisingly, that secretion of any heterologous S-layer proteins, including recombinant S-layer proteins, into the periplasmic space of a Gram-negative host cell or even secretion into the medium surrounding the host cell is possible. This entails the S-layer protein being formed in the periplasm of the host cell not in the form of unordered inclusion bodies but, unexpectedly, in the form of ordered monomolecular layers. In addition, anchoring of heterologous S-layer proteins in the outer or the cytoplasmic membrane of Gram-negative host cells is possible.
S-layer proteins can also be expressed in functional form in eukaryotic cells such as, for example, mammalian cells or yeast. Glycosylation takes place in the case of recombinant S-layer proteins having a eukaryotic fusion portion. In addition, glcosylation may take place in the S-layer protein portion itself.
The process according to the invention makes it possible preferably to express S-layer genes derived from B.stearothermophilus PV72,in particular to express the S-layer genes sbsA and sbsB. In addition, however, it is also possible to express S-layer genes from other organisms (cf., for example, Peyret et al., (1993) supra) by the process according to the invention.
The nucleotide sequence of the gene coding for the mature SbsA protein is indicated in SEQ ID NO. 1 from position 91-3684. The relevant amino acid sequence is depicted in SEQ ID NO. 2. The nucleotide sequence for the gene coding for the mature SbsB protein is indicated in SEQ ID NO. 5 from position 94-2763. The a relevant amino acid sequence is depicted in SEQ ID NO. 6.
In a first preferred embodiment (sbsA), the nucleic acid coding for an S-layer protein is selected from
(i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO. 1 from position 91 to 3684,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code, and
(iii) a nucleic acid which comprises a nucleotide sequence hybridizing with the nucleic acids from (i) or/and (ii) under stringent conditions.
In a second preferred embodiment (sbsB), the nucleic acid coding for an S-layer protein is selected from
(i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO. 5 from position 94 to 2763,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code, and
(iii) a nucleic acid which comprises a nucleotide sequence hybridizing with the nucleic acids from (i) or/and (ii) under stringent condi
Lubitz Werner
Resch Stephanie
Carlson Karen Cochrane
Lubitz Werner
Mitra Rita
Rothwell Figg Ernst & Manbeck
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