Use of gram-positive bacteria to express recombinant proteins

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A novel system for cloning and expression of genes in gram-positive bacteria. The expression system is based on the finding that many gram-positive bacteria sort proteins to their cell surface through cis-acting N-terminal signal sequences and C-terminal anchor regions. In particular, the cell sorting signals of the streptococcal M6 protein, a well-known surface molecule, are used to construct a gram-positive expression system, designated SPEX (Streptococcal Protein Expression). Expression is achieved by cloning the gene of interest into an appropriate SPEX cassette which is then stably introduced into a bacterial host, such as the human commensal Streptococcus gordonii. Depending on the SPEX vector used, recombinant proteins can be anchored to the cell wall prior to release by specific endoproteolytic cleavage or secreted into the culture medium during bacterial growth. The use of host bacteria lacking extracellular proteases should protect secreted proteins from proteolytic degradation. Several expression vectors in this system also produce specifically-tagged recombinant proteins which allows for a one-step purification of the resulting product.

The present invention relates to the construction and use of a novel gram-positive expression vector system. Protein fusions containing the amino and carboxy sorting sequences of a gram-positive surface polypeptide can be anchored to the surface of a heterologous host. Alternatively, recombinant proteins may be secreted into the growth medium. This system can be used for overproducing and purifying recombinant proteins or peptides for any purpose, including but not limited to commercial scale production of diagnostic and vaccine antigens and therapeutic proteins, such as hormones and growth factors.

The ability to overproduce many prokaryotic and eukaryotic proteins has been made possible through the use of recombinant DNA technology. The introduction of chimeric DNA molecules into Escherichia coli has been the method of choice to express a variety of gene products. The main impetus behind the use of E. coli-based protein production systems is the host's short generation time and well-developed genetics. Yet despite the development of many efficient E. coli-based gene expression systems in recent years, the most important concern continues to be that associated with downstream processing of the product. Recombinant proteins produced in E. coli do not readily cross the outer cell membrane (OM); as a result, polypeptides must be purified from the cytoplasm or periplasmic space (PS). Purification of proteins from these cellular compartments can be somewhat difficult. Frequently encountered problems include low product yields, contamination with potentially toxic cellular material and the formation of large amounts of partially folded polypeptide chains in non-active aggregates, called inclusion bodies. As a result of these inherent purification difficulties, a great deal of attention has recently been focused on the natural ability of many gram-positive bacteria to export proteins beyond their cell wall boundaries. In spite of the fact that the genetics of gram-positive bacteria are not as well-elucidated as those of E. coli, these microorganisms have, nevertheless, become recognized as more favorable candidates as hosts for the production of recombinant proteins.

Proteins exported across the gram-positive cytoplasmic membrane (CM) generally have two fates: they are either released (secreted) into the extracellular milieu or they remain anchored to the cell wall/membrane. Many of the recently developed gram-positive based expression systems have relied exclusively on the former route of protein export (i.e., secretion). The basic strategy for directing the secretion of proteins in gram-positive expression systems involves fusing target proteins with functional N-terminal signal sequences of the gram-positive secretion systems currently available. By far, the most popular hosts belong to the genus Bacillus. This group of microorganisms has been used by industry for the production of a variety of economically important proteins; Glenn, Ann. Rev. Microbiol., 30:41. Perhaps the most extensively studied gram-positive host-vector expression system is based on the B. amyloliquefaciens alpha-amylase gene; Palva et al., Proc. Natl. Acad. Sci. U.S.A., 79:5582 (1982); Palva et al., Gene, 22:229 (1983); Lundstrom et al., Virus Res., 2:69. However, expression systems based on other gram-positive hosts such as B. subtilis, B. coagulans, B. licheniformis, Lactococcus and Lactobacillus spp., Staphylococcus spp., Streptomyces spp. and Corynebacterium spp. have also been developed for general use.

Gram-positive secretion systems provide several advantages over traditional E. coli based expression systems. One obvious benefit is that proteins exported beyond the cell wall usually retain their native conformation. As a result, one can take full advantage of established purification protocols which are based on the functional properties of the active protein. In addition, gram-positive expression systems usually generate higher protein yields which are generally free of potentially toxic contaminating cellular material.

There are, however, several practical limitations to the purification of extracellular proteins. The first concern is that of protein instability. Recombinant proteins secreted by many gram-positive hosts are extremely sensitive to proteolytic degradation by host-encoded extracellular proteases. The presence of such proteases can drastically affect protein yield. Fortunately, significant improvements have been achieved in maintaining the viability of secreted proteins by genetically modifying host strains with reduced extracellular protease activities.

Another important point to consider when using gram-positive secretion systems is the requirement for extensive downstream processing. It is widely accepted that the purification of recombinant proteins from bulky, large volume fermentations is an extremely time consuming and costly proposition, both in terms of equipment and manpower. In those cases where the use of a gram-positive secretion system is simply not practical, it is common to resort to a less expensive, gram-negative based expression system. The decision to use E. Coli as an alternative expression host, despite the potential pitfalls, is made simply on the basis that recombinant polypeptides remain associated with the bacterial cell (i.e., intracellular or periplasmic) and hence are generally easier to purify. Therefore, it is clear that what is needed in this art is a bacterial expression system that incorporates the salient features of both gram-positive (i.e., protein secretion) and gram-negative (i.e., protein compartmentalization) systems. A novel, but simple alternative to the currently available bacterial expression systems would be the development of a gram-positive system that is able to specifically anchor or attach recombinant proteins directly to the cell wall surface. In this way the anchoring process can become an integral part of the purification process. For example, cells harboring a recombinant protein attached to the cell surface would be washed, collected, resuspended in a small volume and then treated with a specific agent to affect the release of the desired protein. Following removal of the bacterial cells, the resulting supernatant fluid would be highly enriched for the protein product.

The cell wall of gram-positive bacteria is a complex organelle, which is assembled from peptidoglycans, carbohydrates, and proteins with different biological properties. Surface proteins differ from naturally secreted products in that the former require specific sorting signals that presumably allow them to deviate from the normal default pathway of protein export. Despite the fact that many of the biologically important gram-positive proteins, including the streptococcal M protein, and the protein A and fibronectin binding proteins of S. aureus, are anchored at the cell surface, the cell wall of gram-positive bacteria remains a relatively unknown cellular compartment.