Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-07-08
1998-07-14
Feisee, Lila
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4353201, 435 698, 4352523, 4352525, 435 711, 435 712, 43525231, 43525411, 935 29, 935 27, 935 48, 935 72, 935 74, C12P 2100, C12N 121, C12N 1500, C12N 1532
Patent
active
057802610
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method and expression system for enhanced production of industrially and medically important exoproteins in gram-positive bacteria, especially species of the genus Bacillus.
BACKGROUND
In gram-positive bacteria secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external medium. On the other hand, gram-negative bacteria are surrounded by two cell (or surface) membranes; they have no cell wall. In gram-negative bacteria, most exported proteins are not released from the cell but stay in the inter-membrane periplasmic space and in the outer membrane.
Two types of components of the secretion machinery have been identified in E. coli: soluble cytoplasmic proteins and membrane associated proteins (see for review, Wickner et al., (1991) Annu. Rev. Biochem., 60:101-124). Soluble cytoplasmic proteins, including SecB and heat shock proteins, all prevent the folding of precursors of secreted proteins into a conformation incompatible with secretion. The set of membrane-associated proteins includes the peripheral membrane protein SecA, integral membrane proteins SecY, SecE, SecD, SecF and the signal peptidases Lep and Lsp (reviewed in Hayashi, S. and Wu, H. C. (1990) J. Bioenerg. Biomembr., 22:451-471; Dalbey, R. E. (1991) Mol. Microbiol., 5:2855-2860). These membrane-associated proteins are involved in binding of the precursor and in its translocation across the cytoplasmic membrane, followed by cleavage of the signal peptide and release of the protein.
Knowledge on the secretion machinery of gram-positive bacteria is more limited. The available data on B. subtilis, the genetically and physiologically well characterized model organism of the genus, suggest an overall similarity with that of E. coli, but also differences in the structure and specificity of individual components, possibly reflecting demands set by the very different composition and architecture of the respective cell envelopes.
Gram-positive bacteria such as B. subtilis, B. amyloliquefaciens, B. licheniformis have a very high capacity for secreting proteins, and indeed, many bacillar extracellular enzymes are utilized industrially. Since secreted proteins in gram-positive bacteria are so important commercially, and since the gram-positive secreted proteins traverse through a cell envelope with a very different structure from that of E. coli the molecular mechanisms of protein secretion in gram-positive bacteria is of considerable academic and industrial importance.
In this regard we recently discovered a novel component, the PrsA protein, of the secretion machinery of B. subtilis (Kontinen, V.P. and Sarvas, M., (1988) J. Gen. MicrobioL, 134:2333-2344; Kontinen, V.P., et al., (1991) Mol. Microbiol. 5:1273-1283). The prsA gene, which encodes the PrsA protein, was initially defined by nonlethal mutations that decreased the secretion of several exoproteins (Kontinen, V. P. and Sarvas, M., (1988) J. Gen. Microbiol., 134:2333-2344). Based on the DNA sequence of the cloned prsA gene and our subsequent work with this gene and protein, we assert that prsA encodes a protein (PrsA) that acts as a chaperone, and is translocated across the cytoplasmic membrane (for the initial work, see Kontinen, V. P., etal., (1991) Mol. Microbiol. 5:1273-1283). The PrsA protein has been found to possess a limited amount of sequence homology (about 30%) with the PrtM protein of Lactococcus lactis, a protein proposed to assist the maturation of an exported serine protease (Haandrikman, A. J., et al, (1989) J. Bacteriol., 171:2789-2794; Vos, P., et al., (1989) J. Bacteriol., 171:2795-2802). A similar function has not been associated with other known proteins of the secretion machinery of bacteria, suggesting that PrsA protein is a novel type of component in the pathway of protein secretion facilitating the release and probably folding of secreted proteins after their translocation across the cytoplasmic membrane in gram-positive bacteria.
It is advantageous to produce
REFERENCES:
patent: 4711843 (1987-12-01), Chang
patent: 4879213 (1989-11-01), Fox et al.
patent: 4952508 (1990-08-01), Chang et al.
patent: 4965197 (1990-10-01), Liebel et al.
patent: 4994379 (1991-02-01), Chang et al.
Lazar et al (1988) Mol. J. Cell. Biol. vol. 8(3), 1247-1252.
Burgen et al (1990) J. Cell. Biol. vol. 111, 2129-2138.
Wickner, W., Driessen, A.J.M., and Hartl, F. The Enzymology of Protein Translocation Across the Escherichia coli Plasma membrane. Annual Reviews of Biochemistry 60: 101-124 (1989).
Kontinen, V. and Sarvas, M. Mutants of Bacillus subtilis Defective in Protein Export. Journal of General Microbiology 134: 2333-2344 (1988).
Kontinen, V. and Sarvas, M. A Gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export. Molecular Microbiology 5(5): 1273-1283 (1991).
Haandrikman, A.J., et al. Identification of a Gene Required for Maturation of an Extracellular Lactococcal Serine Proteinase. Journal of Bacteriology 171(5): 2789-2794 (1989).
Vos, P. et al. A Maturation Protein is Essential for Production o Active Forms of Lactococcus lactis SK11 Serine Proteinase Located in or Secreted from the Cell Envelope. Journal of Bacteriology 171(5): 2795-2802 (1989).
Ferrari, F.A., Nguyen, A., Lang, D. and Hoch, J.A. Construction and Properties of an Integrable Plasmid for Bacillus subtilis. Journal of Bacteriology 154(3): 1513-1515 (1983).
Sibakov, M., Sarvas, M. and Palva, I. Increased secretion of .alpha.-amylase from Bacillus subtilis caused by multiple copies of .alpha.-amylase Gene from B. amyloliquefaciens is not further increased by genes enhancing the basic level of secretion. FEMS Microbiology Letters 17:81-85 (1983).
Gardel, C., Johnson, K., Jacq, A. and Beckwith, J. The secD locus of E. coli codes for two membrane proteins required for protein export. EMBO Journal 9(10): 3209-3216 (1990).
Kontinen Vesa
Sarvas Matti
Feisee Lila
Masood Khalid
Meyer, Esq. Virginia H.
The Finnish National Public Health Institute (KTL)
LandOfFree
Method and system for enhanced production of commercially import does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and system for enhanced production of commercially import, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and system for enhanced production of commercially import will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1880743