Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2001-07-30
2003-08-26
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S005000, C435S007200, C435S069100, C424S184100, C424S234100
Reexamination Certificate
active
06610517
ABSTRACT:
The present invention relates to host cells which contain at least two functional recombinant polypeptides, at least one of which is bound to a support, preferably in each case in different cell compartments, e.g. cytosol, cytoplasmic membrane, periplasm and outer membrane, and also to methods for preparing said host cells. The cells of the invention are particularly suited as bioreactors for carrying out enzymatic reaction cascades for which compartmentalization of individual enzymes is advantageous or necessary.
The use of living cells as “enzyme reactors” for preparing biological substances, e.g. polyhydroxy fatty acids, is of great importance for the biotechnological industry. To this end, it is common to introduce foreign genes into a host cell and to express said genes, in order to obtain in this manner a host cell with recombinant enzymes, which is capable of synthesizing a desired product. A disadvantage of known methods, however, was that the enzymes generated in the host cells by expression of the foreign genes were not stable, had too little activity or were present in too small an amount. Particular difficulties also stepped when carrying out multistage reactions in which substrates or products of one stage may have an adverse effect on other stages.
It was an object of the present invention to eliminate at least partially the problems of the prior art and to provide host cells which are capable of presenting functional recombinant polypeptides in a stable form and, in particular, of carrying out multistage enzyme reactions.
The object is achieved by providing a host cell comprising at least two functional recombinant polypeptides at least one of which is bound to a support.
Surprisingly, it was found that support-bound recombinant expression of heterologous polypeptides preferably in different compartments of a cell, e.g. of Gram-negative bacterial cells or of eukaryotic cells, leads to stable presentation of the heterologous polypeptides in a functional (i.e. immunologically or/and biologically, e.g. enzymatically, active) form. If the host cell is a Gram-negative bacterial cell, the cell compartments may preferably be selected from the cytosol, the cytoplasmic membrane (outside and inside), the periplasmic space and the outer membrane (outside and inside). If the host cell is a eukaryotic cell, said compartments may preferably be selected from the cytosol, the cytoplasmic membrane (outside and inside) and cell organelles such as, for example, Golgi, lysosomes, mitochondria, chloroplasts, vacuoles or endoplasmic reticulum.
The functional recombinant proteins present in the host cell are preferably cooperative, i.e. they fulfill a common immunological or/and biological function, for example as enzymes in a multistage reaction cascade.
At least one, preferably a plurality, of the functional recombinant polypeptides is bound to a support, for example in the form of fusion polypeptides, and contain at least one functional domain and at least one support domain. Preferred forms of support-bound polypeptides are S-layer structures (fusion polypeptides with S-layer support domains), membrane-bound polypeptides (fusion polypeptides with membrane-integrated support domains) or/and components of recombinant phage structures. If it is desired to export the functional polypeptides from the cytosol to other cell compartments, said polypeptides are expressed together with suitable targeting domains which facilitate export to the cell compartment desired in each case. Examples of targeting domains are signalpeptide or/and helper sequences which facilitate passage through the membranes.
In a preferred embodiment of the invention, at least one of the support-bound polypeptides is present as recombinant S-layer structure. S-layers are crystalline bacterial cell surface proteins which are composed of identical self-assembled units. Genetic data and sequence information for various S-layer genes from microorganisms can be formed, for example, in Peyret et al. (Mol. Microbiol. 9 (1993), 97-109). These data are expressly incorporated by reference.
Preferred S-layer genes are the
B.stearothermophilus
PV72 genes sbsA and sbsB. The sequences of these genes can be found, for example, in the international patent application PCT/EP97/00432 which also discloses production of a recombinant S-layer fusion protein in the cytoplasm of Gram-negative host cells. The international patent application PCT/EP98/04723 in turn describes production of a recombinant S-layer protein in various compartments of Gram-negative bacteria cells or eukaryotic cells. Regarding the construction of recombinant S-layer genes and production of suitable expression constructs, these two said international applications are expressly referred to. However, no indication of coexpression of two different functional recombinant polypeptides is found there.
Surprisingly, it was found that it is possible to co-express simultaneously or/and sequentially a plurality of recombinant S-layer proteins, where appropriate in combination with further heterologous proteins, for example in various compartments of host cells, in particular of Gram-negative bacterial cells and eukaryotic cells.
The nucleotide sequence of the gene coding for the mature SbsA protein is indicated from position 91-3684 in SEQ ID No. 1. The corresponding amino acid sequence is depicted in SEQ ID No. 2. The nucleotide sequence of the gene coding for the mature SbsB protein is indicated from position 94-2763 in SEQ ID No. 3. The corresponding amino acid sequence is depicted in SEQ ID No. 4.
In a first preferred embodiment (sbsA), the nucleic acid coding for the support domain of a functional peptide is selected from
(i) a nucleic acid which comprises the nucleotide sequence from position 91 to 3684 shown in SEQ ID No. 1,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the framework 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 the support domain of a functional peptide is selected from
(i) a nucleic acid which comprises the nucleotide sequence from position 94 to 2763 shown in SEQ ID No. 3,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the framework 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.
“Stringent hybridization” in accordance with the present invention means that hybridization still occurs even after washing at 55° C., preferably 60° C., in an aqueous low-salt buffer (e.g. 0.2×SSC) (see also Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual).
Preferred sites for inserting peptide- or polypeptide-coding sequences into the sbsA gene are regions between positions 200-3600 of the nucleotide sequence shown in SEQ ID No. 1. Particularly preferred insertion sites are the NruI cleavage site at position 585, the PvuII cleavage site at position 881, the SnaB I cleavage site at position 920, the PvuII cleavage site at position 2507 and the PvuII cleavage site at position 2652 (PCT/EP 97/00 432). Further preferred insertion sites are positions 562, 1087, 1813, 1947, 2295, 2652, 3046, 3484 and 3594. The positions indicated in each case refer to the first nucleotide of the insertion.
Preferred sites of insertion into the sbsB gene are regions between positions 200-2600 of the nucleotide sequence shown in SEQ ID No. 3. Particularly preferred insertion sites are positions 410 (codon 136), 484 (codon 161/162) and 1583 (codon 528/529) (PCT/EP 97/00432). Further preferred insertion sites are positions 598, 1012, 1435, 1808 and 2301, the position indicated in each case referring to the first nucleotide of the insertion.
Alternatively or additionally, it is also possible to produce support-bou
Park Hankyel T.
Rothwell Figg Ernst & Manbeck P.C.
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