Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2002-05-17
2004-02-17
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S252300, C435S320100, C536S023700
Reexamination Certificate
active
06692951
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to processes for the production of proteins by micro-organisms. Specifically, it relates to the secretion of heterologous proteins by micro-organisms, in particular by Gram-positive bacteria, especially by the bacterial host Bacillus.
It also relates to (the overexpression of) a novel gene encoding a protein involved in the early stages of prokaryotic protein secretion. Specifically, it relates to the overexpression of said gene within a Bacillus host (over)expressing heterologous proteins.
BACKGROUND OF THE INVENTION
B. subtilis
and (closely) related bacilli secrete proteins directly into the growth medium to high concentrations. Secretion as a mode of production of proteins of interest, be it homologous to the host or heterologous to the host, be it of recombinant origin or not, provides several advantages over intracellular production. It for instance facilitates purification of the product, it theoretically will lead to a higher yield, no aggregation of the product will occur, and it gives the possibility for continuous cultivation and production. However, attempts to secrete heterologous proteins from
B. subtilis
and (closely) related organisms at commercially significant concentrations have, with few exceptions, met with little success.
Nearly all secreted proteins use an amino-terminal protein extension, known as the signalpeptide, which plays a crucial role in the targeting to, and translocation of precursor proteins across the membrane and which is proteolytically removed by a signalpeptidase during or immediately following membrane transfer. The newly synthesized precursor proteins are recognized by specific proteins in the cytoplasm collectively called chaperones. These chaperones prevent polypeptides, destined for translocation, to aggregate or fold prematurely leading to an export incompatible conformation.
For instance, SecB, GroEL/GroES and DnaK/DnaJ are the presently known chaperones in the export pathway of
E. coli
. For the productive binding of precursor proteins to translocation sites in the cytoplasmic membrane SecA is needed. SecA, a protein of which cytoplasmic, peripheral as well as integral membrane forms have been detected, has an ATPase activity which mediates the initial channelling of precursor proteins into the export pathway.
The SecA subunit acts as a receptor recognizing the leader and mature domains of the preproteins (Lill et al. 1990) as well as the SecB chaperone (Hartl et al. 1990). It has been suggested that SecA penetrates into the membrane, after binding of ATP, and so promotes the coinsertion of the preprotein. After hydrolysis of bound ATP the preprotein is released from the SecA protein (Schiebel et al. 1991). Translocation is completed with the proton motive force as the main driving force and requires members of the integral membrane part of the preprotein translocase complex like SecY, SecE and SecG (p12/Band1). SecD and SecF are also integral membrane proteins and are probably participating in the late steps of protein translocation.
For many years, the protein secretion machinery in prokaryotes has been considered to be independent from the protein secretion system found in higher eukaryotes (Luirink et al, 19920. In mammalians, targeting of secretory proteins to the endoplasmic reticulum (ER) is mediated by the signal recognition particle (SRP), which is a ribonucleoprotein particle composed of one RNA molecule (SRP 7S RNA) and six polypeptides of 9, 14, 19, 54, 68 and 72 kD. The SRP proteins are associated with the RNA as either monomers (SRP19 and SRP54) or heterodimers (SRP9/14 and SRP68/72).
As soon as the signal peptide of secreted and transmembrane proteins has emerged from the ribosome, it is recognized and bound by SRP, which also has affinity for the ribosome. This association slows down the elongation of the polypeptide chain (elongation arrest). When the complex of SRP, nascent polypeptide chain, and ribosome bind to the SRP receptor (SR or docking protein) associated with the ER membrane, the nascent polypeptide chain is displaced from SRP in a GTP-dependent reaction and protein translation is resumed.
The translocation of the polypeptide into the ER takes place co-translationally through a protein pore, the translocon (Gilmore et al. 1993). Thus, the SRP functions both as a cytosolic chaperone preventing premature folding of the preprotein by coupling translation to translocation and as a pilot to guide the preprotein to the SRP receptor complex in the membrane. The 54 kD subunit of SRP (SRP54) binds to the signal peptide when it emerges from the ribosome and therefore seems to have a key function in the SRP-mediated process of protein secretion.
To day more and more data become available indicating that an SRP-mediated export pathway may also function in other organisms. Homologues of mammalian SRP components have been isolated from Yeast (Hann et al. 1989),
E. coli
(Bernstein et al. 1989, and Rymisch et al. 1989),
Mycoplasma mycoides
(Samuelsson, 1992) and
Bacillus subtilis
(Struck et al. 1989, and Honda et al. 1993).
So it is likely that an SRP-mediated pathway functions in prokaryotes in a separate secretory pathway or may form part of the general secretory pathway.
In
E. coli
members of an SRP-like secretory pathway were identified. These members are Ffh (Fifty four homologue) and a 4.5S RNA molecule which are homologous to the SRP54 and SRP 7S RNA of eukaryotic SRP (Ribes et al. 1990). It is shown that Ffh interacts specifically with the signal sequence of nascent presecretory proteins (Luirink et al. 1992).
E. coli
protein FtsY, which originally has been implicated in cell division (because its gene is located in an operon together with FtsE and FtsX) displays striking sequence similarity with the subunit of mammalian docking protein. Several observations suggest that FtsY is the functional
E. coli
homologue of the mammalian SRP receptor (Luirink et al. 1994). Depletion of either FtsY, Ffh or the RNA component of the
E. coli
SRP affects the export of several secretory proteins.
Also in
B. subtilis
components of the SRP-like secretory pathway have been found. The Small Cytoplasmic RNA (scRNA) was shown to have a functional relationship with the human SRP 7S RNA and the
E. coli
4.5S RNA (Nakamura et al., 1992). The
B. subtilis
scRNA is transcribed from the scr gene as a 354 nucleotide precursor which is then processed to a 271 nucleotide RNA at the 5′ and 3′ end (Struck et al., 1989), which is similar to its eukaryotic homologue (300 nucleotides) but much larger than the
E. coli
4.5S RNA (114 nucleotides). Also the secundary structure of the scRNA is very similar to the eukaryotic SRP 7S RNA, lacking only the domain III (Struck and Erdmann, 1990). This is in contrast to the other eubacterial SRP-like RNAs, which only fold into a single hairpin corresponding to domain IV (Poritz et al., 1988). Therefore the
B. subtilis
scRNA is both in size and secundary structure an intermediate between prokaryotic and eukaryotic SRP-like RNA.
Besides the scr gene another gene encoding a SRP constituent has been isolated from
B. subtilis
. The ffh gene was found to encode the Ffh protein which shows homology to both the
E. coli
and eukaryotic SRP54 protein (Honda et al., 1993).
It is not unlikely that chaperones or members of the SRP-like secretion pathway may become a rate-limiting step in the secretion pathway, the result of which being that the majority of the heterologous protein expressed will aggregate or fold prematurely. This effect could be the reason why attempts to secrete heterologous proteins in high amounts from Gram-positive micro-organisms, in particular
B. subtilis
and (closely) related micro-organisms have met with little success. Overexpression of particular members of the
B. subtilis
secretion machinery, especially of chaperone-like proteins which are the rate-limiting step in the secretion pathway would solve this problem. It is to be understood that the terms “chaperone” and “secretion factor” are not completely
Broekhuizen Cornelis P.
Kerkman Richard
Quax Wilhelmus J.
Genencor International Inc.
Genencor International Inc.
Slobodyansky Elizabeth
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