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  Co-expression of recombinant proteinsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Recombinant Dna Technique Included In Method Of Making A Protein Or Polypeptide, AntigensThe Patent Description & Claims data below is from USPTO Patent Application 20070031937. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to the fields of molecular biology, biochemistry and vaccinology, in particular, to the co-expression of recombinant proteins. BACKGROUND TO THE INVENTION [0002] Recombinant proteins expressed from E. Coli, are often made as insoluble inclusion bodies. While the purification of inclusion bodies is relatively straightforward and can lead to a >90% purification of the recombinant protein, the resulting protein is often denatured and may be biologically inactive. In some instances, it may be advantageous to overproduce a recombinant protein in a soluble form. Recombinant proteins can also be degraded by host proteases. Expression of recombinant proteins in the presence of particular proteins, such as potential molecular chaperones, may have the effect of protecting them from degradation and ensure correct folding. In other instances, it may be useful to produce two vaccine components, from different organisms, in the same production cycle. Co-expression of recombinant proteins encoded on genes from multiple organisms can lead to improved production time and costs. [0003] Otitis media is the most common illness of early childhood, with 60 to 70% of all children of less than 2 years of age experiencing between one and three ear infections (ref. 1, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure). Chronic otitis media is responsible for hearing, speech and cognitive impairments in children. In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and the insertion of tympanostomy tubes. It is estimated that an additional $30 billion is spent per annum on adjunct therapies, such as speech therapy and special education classes. The disease is caused by bacterial and/or viral infections, and many of the bacteria are becoming antibiotic resistant. Infection with Streptococcus pneumoniae accounts for about 50% of bacterial disease, while non-typeable Haemophilus influenzae (NTHi) infections account for about 30%, and Moraxella catarrhalis is responsible for about 20% of acute otitis media. An effective prophylactic vaccine against otitis media is thus desirable. [0004] When under environmental stress, such as high temperature, organisms overproduce stress response or heat shock proteins (hsps). In some instances, hsps have also been demonstrated to be molecular chaperones (ref. 2). The bacterial HtrA or DegP heat shock proteins are expressed under conditions of stress and the H. influenzae HtrA protein has been shown to be a partially protective antigen in the intrabulla challenge model of otitis media (ref. 3). The HtrA proteins are serine proteases and their proteolytic activity makes them unstable. In addition, as components of a multi-component vaccine, the wild-type HtrA protein degrade admixed antigens. The site-directed mutagenesis of the H. influenzae htrA gene (termed hin47) and the properties of the mutants have been fully described in U.S. Pat. No. 5,506,139 (Loosmore et al.), assigned to the assignee hereof and the disclosure of which is incorporated herein by reference. The non-proteolytic HtrA analogue, H91A Hin47, has been shown to be a protective antigen in the intrabulla chinchilla model of otitis media (ref. 3). The mature H91A Hin47 protein is produced at 40 to 50% of total E. coli protein, in a soluble form. It may also be produced with its leader sequence, at a level of 20 to 30% of total E. coli protein. In this form, it may function as a molecular chaperone, anchored in the periplasmic membrane (ref. 4). [0005] During natural infection by NTHi, surface-exposed outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and/or protective antibodies and are therefore potential vaccine candidates. Barenkamp and Bodor (ref. 5) demonstrated that convalescent sera from children suffering from otitis media due to NTHi, contained antibodies to high molecular weight (HMW) proteins. About 70 to 75% of NTHi strains express the HMW proteins, and most of these strains contain two gene clusters termed hmw11ABC and hmw2ABC (refs. 6, 7). The HMWA proteins have been demonstrated to be adhesins mediating attachment to human epithelial cells (ref. 8). Immunization with a mixture of native HMW1A and HMW2A proteins resulted in partial protection in the chinchilla intrabulla challenge model of otitis media (ref. 9). The production yields of native HMW proteins from H. influenzae strains are very low, but a method for producing protective recombinant HMW (rHMW) proteins has been described in copending U.S. patent application Ser. No. 09/167,568 filed Oct. 7, 1998 (WO 00/20609), assigned to the assignee hereof and the disclosure of which is incorporated herein by reference. The HMWB and HMWC proteins are thought to function as molecular chaperones, responsible for the correct processing and secretion of the HMWA proteins (ref. 10). [0006] A second family of high molecular weight adhesion proteins has been identified in about 25% of NTHI and in encapsulated H. influenzae strains (refs. 11, 12, 13). The NTHi member of this second family is termed Haemophilus influenzae adhesin or Hia, and the homologous protein found in encapsulated strains is termed Haemophilus influenzae surface fibril protein or Hsf. The hia gene was originally cloned from an expression library using convalescent sera from an otitis media patient, which indicates that it is an important immunogen during disease. Production of the full-length recombinant Hia protein in E. coli appears to be toxic to the host, so a series of N-terminally truncated proteins was made as described in copending U.S. patent application Ser. No. 09/268,347 filed Mar. 16, 1999 and in PCT Patent Application No. PCT/CA00/00289 filed Mar. 16, 2000, both assigned to the assignee hereof and the disclosures of which are incorporated herein by reference. The V38 rHia protein was chosen for further development as a vaccine, but it was found that the first 6 amino acids of this protein were deleted from a portion of the product during synthesis in E. coli, leading to a mixture of V38 rHia and S44 rHia. When an expression construct was developed to produce the S44 rHia, it was found that the N-terminus was stable, with only S44 rHia product being made. The rHia products appear as a doublet on SDS-PAGE when expressed alone. However, when co-expressed with H91A Hin47, the S44 rHia is produced as a single band, as described below. [0007] The S. pneumoniae antigen, pneumococcal surface adhesin A or PsaA, is a protective antigen in an animal model (ref 14), which is produced in high yield from E. coli as a 37 kDa protein. The protein may be produced as a lipoprotein if the psaA gene contains a sequence encoding a lipoprotein leader sequence, or as a soluble protein if the gene encodes the mature protein. The protein and the encoding nucleotide sequence are described in U.S. Pat. No. 5,854,466, the disclosure of which is incorporated herein by reference. When co-expressed with H91A Hin47, both proteins are produced in high yield, as described below. They may be separated by hydroxylapatite (HTP) column chromatography during purification, resulting in the high level production of two vaccine components from different organisms, as described herein. [0008] The over-production of E. coli chaperone proteins DnaK, DnaJ and GrpG (Hsp70) or GroEL and GroES (Hsp60) results in increased solubility of recombinant human protein kinases Csk, Fyn or Lck (ref. 15). These chaperones have also been shown to aid in the refolding of an allergen (Japanese cedar pollen) in E. coli (ref. 16). The E. coli Skp chaperone has also been used to increase the solubility of recombinant single-chain antibody fragments when co-expressed in E. coli (ref. 17). All these systems use a native E. coli chaperone to aid in the solubility and folding of recombinant proteins in E. coli. The present invention, for the first time, uses a heterologous protein as the chaperone SUMMARY OF THE INVENTION [0009] The present invention is directed to the expression of recombinant protein and expression vectors for utilization therein. In one feature of the present invention, such expression is effected in conjunction with expression of non-proteolytic analogs of Haemophilus Hin47 and in another feature of the present invention, such expression is effected in conjunction with expression of a high molecular weight protein of non-typeable Haemophilus which is hmwBC, hmwB or hmwC. [0010] Accordingly, in one aspect of the present invention, there is provided an expression vector, comprising a nucleic acid molecule encoding a non-proteolytic analog of a Hin47 protein of a strain of Haemophilus including a portion thereof encoding the leader sequence for said non-proteolytic analog, and a promoter operatively connected to said nucleic acid molecule to direct expression of said non-proteolytic analog of a Hin47 protein having said leader sequence in a host cell. [0011] In the various aspects of the invention involving a non-proteolytic analog of Hin47 protein, such analog may be a mutation of natural Hin47 protein in which at least one amino acid selected from the group consisting of amino acids 91, 121 and 195 to 201 of natural Hin47 protein has been deleted or replaced by another amino acid. Preferably, the analog has histidine 91 replaced by alanine. This specific analog is termed H91A Hin47. [0012] The vector may be a plasmid vector which may be one having the identifying characteristics of plasmid JB-3120-2 as seen in FIG. 1A, such identifying characteristics being the nucleic acid sequences and restriction sites identified therein. [0013] In accordance with another aspect of the present invention, there is provided an expression vector for expression of a recombinant protein in a host cell, comprising a nucleic acid molecule encoding a non-proteolytic analog of a Haemophilus Hin47 protein, at least one additional nucleic acid molecule encoding a recombinant protein, and at least one regulatory element operatively connected to said first nucleic acid molecule and said at least one additional nucleic acid molecule to effect expression of at least said recombinant protein in the host cell. [0014] In one embodiment, the nucleic acid molecule encoding the non-proteolytic analog of a Hin47 protein includes a portion thereof encoding the leader sequence of the non-proteolytic analog, or such portion may be absent. [0015] The at least one additional nucleic acid molecule may encode a Hia or Hsf protein of a strain of Haemophilus influenzae, specifically a Hia protein which is N-terminally truncated. The N-terminal truncation may be S44 or V38. The construction of such N-terminal truncations is described below. [0016] The vector may be a plasmid vector, which may be one having the identifying characteristics of plasmid DS-2542-2-2 as seen in FIG. 5 or of plasmid JB-3145-1 seen in FIG. 10, for expression of N-terminally truncated Hia proteins. Such identifying characteristics are the nucleic acid sequences and restriction sites seen in the respective Figures. [0017] The at least one additional nucleic acid molecule may encode a PsaA protein of a strain of Streptococcus pneumoniae. The vector may be a plasmid vector having the identifying characteristics of plasmid JB-3073R-1 as seen in FIG. 12 or of plasmid JB-3090-1 or JB-3090-7, as seen in FIG. 13, and expressing the PsaA protein. Such identifying characteristics are the nucleic acid sequences and restriction sites seen in the respective Figures. [0018] The expression vector provided in this aspect of the present invention may be utilized in the generation of a recombinant protein by expression from a suitable host cell, such as E. coli. Accordingly, in another aspect of the present invention, there is provided a method for expressing at least one protein, which comprises providing a first nucleic acid molecule encoding a non-proteolytic analog of a Hin47 protein of Haemophilus; [0019] isolating at least one additional nucleic acid molecule encoding a protein other than Hin47; introducing the first nucleic acid molecule and the at least one additional nucleic acid molecule into a cell to produce a transformed cell; and growing the transformed cell to produce at least one protein. The nucleic acid molecules and regulatory elements may be incorporated into any of the specific expression vectors discussed above. [0020] As noted above, one feature of the present invention involves vectors based on nucleic acid encoding high molecular weight (HMW) protein of a non-typeable strain of Haemophilus. Accordingly, in accordance with a further aspect of the present invention, there is provided an expression vector, comprising a nucleic acid molecule encoding a high molecular weight protein of a non-typeable strain of Haemophilus selected from the group consisting of hmwB and hmwC, and a promoter operatively connected to said nucleic acid molecule to direct expression of said high molecular weight protein in a host cell. [0021] The vector may be a plasmid vector which may have the identifying characteristics of IN-137-1-16 shown in FIG. 18A or of pT7 hmwC shown in FIG. 19A, such identifying characteristics being the nucleic acid sequences and restrictions sites identified in the respective Figures. Continue reading... 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