The large-scale production of proteins as the result of the use of recombinant DNA technology in bacteria faces several difficulties including: (1) overgrowth of the culture by revertant bateria and bacteria that have lost the recombinant DNA plasmid due to genetic instability of the transformed strain; (2) difficulty in recovering the desired protein products in an active form due to the formation of inclusion bodies; and (3) difficulties in recovering the protein product without contaminants due to the necessity of having to lyse the bacteria in order to isolate the product.
Actively growing bacteria containing recombinant DNA plasmids grow more slowly than those without the recombinant DNA plasmids when the proteins coded by the recombinant DNA are being actively manufactured. Therefore, large quantities of bacteria can be difficult to produce without potential problems of plasmid instability. If a plasmid gene is expressed continuously, a high proportion of the plasmid protein is produced by the bacterium as:the product. However, because the bacterium has diverted substantial resources away from its own growth, the bacterium grows more slowly than it otherwise would.
Further, some bacteria may spontaneously lose the plasmid and revert to the parental form which grows more rapidly. For example, a bacterium containing free plasmids can lose the plasmids at a rate of 1 out of 10.sup.7 or 10.sup.6 divisions or even more frequently than one out of 10.sup.6 divisions. (See, for example, L. C. Klotz, Ann. N.Y. Acad. Sci., 369 p.1 (1983)). Therefore, the more rapidly growing, but non-productive, revertant bacteria soon displace the plasmid-bearing bacteria. The problem of plasmid or vector loss becomes increasingly important as the scale of operation increases, making continuous culture processes practically impossible for large-scale batch cultures.
Additionally, Escherichia coli (hereinafter E. coli) is presently the organism of choice for genetic manipulation. A wide range of vectors, promoters, etc., and a detailed knowledge of the genetic system and physiology allow for elegant genetic manipulation of E. coli. However, E. coli and other gram-negative bacteria do not normally excrete the proteins produced from the cells, and, therefore, the bacterial cells must be ruptured to obtain the product of interest. Consequently, the product must be recovered from an aqueous solution containing many macromolecules in addition to the desired product. In particular, recovery of a product sufficiently free of contaminants (such as, for example, endotoxins) to be useful in therapeutic or food applications is difficult. Also, resolubilizing the isolated proteins in an active form is often extremely difficult because proteins formed from recombinant DNA molecules which remain within the cell form inclusion bodies.
One method which is known for synthesizing within a bacterial host, and excreting through the membrane of the host, a selected protein or polypeptide is disclosed in U.S. Pat. No. 4,338,397 and European Patent Application 0,038,182 A2 (1981). The method involves forming a cloning vehicle wherein a non-bacterial DNA fragment which codes for the precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide, is inserted behind a promoter of either a bacterial or phage gene within a cloning vehicle or a DNA fragment of the bacterial or phage gene. The method produces mature proteins or polypeptides free of signal sequences or other chemical substituents. However, the majority of the selected protein or polypeptide, i.e. as much as 90%, is found in the periplasmic space of the host cells, rather than being secreted beyond the host cell membrane into the culture medium. Further, these patent documents disclose that the bacterial host cells must be actively growing, and, therefore, cannot be immobilized without the attendant difficulties of the bacterial cells dividing and thereby possibly clogging the protein production system.
U.S. Pat. No. 4,336,336 discloses a fused gene for producing excreted proteins in bacteria. The fused gene is created by fusing a gene for a cytoplasmic protein to a gene for a non-cytoplasmic protein. However, when gram-negative bacteria are used, the genes for the non-cytoplasmic protein are those coding for proteins that travel to the cell surface or periplasmic space and, therefore, the proteins thus produced are not actually excreted from the cell. Further, the bacteria must be actively growing in order to produce the protein, and, the protein thus produced contains an NH.sub.2 -terminus coded for by the carrier protein.
PCT Application WO 80/00030 also describes a method for producing excreted proteins from a fused gene. According to the disclosure, a plasmid is constructed which contains a DNA sequence coding for a selected protein from a eukaryotic cell inserted into a cleaved gene representing a periplasmic or extracellular bacterial protein. only secretion of the protein into the periplasmic space is actually demonstrated for E. coli and not excretion into the medium. Further, continuous protein production is neither taught nor suggested.
European Patent Application 0 036 259 A2 (1981) discloses a method and vector for producing a gene product which, if produced in gram-positive bacteria, can be recovered from the growth medium. The gene for the protein to be produced is under the control of an operator, promoter, and ribosomal binding site sequence. Further, the protein to be produced is under the control of a transport mechanism by which the protein is secreted by the host strain. The method is applicable to gram-negative bacteria. However, when gram-negative bacteria are used, the protein is secreted into the periplasmic space instead of the growth medium.
United Kingdom Patent Application 2 091 269 A discloses various plasmid cloning vehicles for the expression of exogenous genes in transforming bacterial hosts. The cloning vehicles comprise a DNA insert fragment coding for the desired polypeptide linked in reading phase with one or more functional fragments derived from an outer membrane protein gene of a gram-negative bacterium. The polypeptide thus produced is expressed with a leader sequence located at the amino terminal such that the desired product is secreted through the cytoplasmic membrane into the periplasmic space or into the cell's outer membrane. Further, it is suggested that the use of a host cell known to be "leaky" may be desirable in that proteins secreted across the cytoplasmic membrane of such cells may ultimately "leak" out into the culture medium through the outer membrane of the cell. However, this patent document does not in any way teach or suggest the possibility of continuous protein production.
It is also known that the addition of various antibiotics to the growth medium of Bacillus subtilis cells carrying plasmids coding for rat proinsulin can inhibit bacterial cell division while allowing continued protein synthesis and excretion of the proteins, even when the microorganisms are immobilized in various support materials including alginate, polyacrylamide and agarose supports. Additionally, an immobilized genetically engineered strain of a periplasmic leaky mutant of E. coli is known to maintain protein synthesis and excrete the protein into the medium even though cell growth has been arrested by addition of nalidixic acid to the growth medium. Mosbach, K., et al, "Formation of Proinsulin by Immobilized Bacillus Subtilis", Nature, 302, 543 (April 1983). However, this system does not solve the problem of plasmid or vector loss while culturing the bacteria to obtain large quantities of transformed cells. Additionally, the method described in Mosbach et al, supra, is disadvantageous in that antibiotics have undesirable side reactions at higher concentrations. Most antibiotics targeted to stop DNA replication will, at sufficient concentration, also affect RNA transcription and consequently protein production. Further, antibiotics are expensive and the high costs would limit the technique of Mosbach et al, supra, to high-value, small-volume protein products.
In addition to the above described methods, several other patent documents are directed to immobilizing cells in a resting or growing state or in a non-viable form as carriers for enzymes. (See for example U.S. Pat. No. 4,347,320, German OLS 28 35 874 A1 (1980) and 28 35 875 A1 (1980)). However, none of these patent documents teaches or discloses the immobilization of L-forms, spheroplasts, leaky mutants, or other forms in which the cell envelope has been altered to allow protein excretion. Further, none of these patents disclose cells which produce proteins.
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