Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1997-02-03
2004-06-08
Duffy, Patricia A. (Department: 1817)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C530S350000
Reexamination Certificate
active
06746859
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the cloning and expression of enterokinase activity and to methods of its making and use.
BACKGROUND OF THE INVENTION
The use of fusion proteins as a tool for recombinant protein production is well known in the biopharmaceutical industry. Fusing the coding sequence for a desired recombinant protein to that of a well-expressed gene has several advantages. Most fusion protein strategies position the protein of interest at the C-terminal end of the highly expressed fusion partner which allows translation initiation to occur on a “proven” gene sequence that is known to be well translated and can help ensure high expression levels. Some fusion partners can confer many advantageous attributes to the fusion protein, such as specific cellular localization, binding to affinity ligands to aid in purification and detection, and even proteolytic and conformational stability.
While fusion proteins offer numerous advantages, this beneficial physical association of the protein domains can also be problematic when it becomes necessary to separate the two (or more) components from their covalent tethering. The method of protein cleavage must be both specific and efficient and must not yield unwanted side products. This is particularly so when utilizing a fusion protein approach for the production of biopharmaceuticals destined for human use. Ideally, the most useful method allows for cleavage at a specific target sequence without regard for the internal protein sequence and/or without regard for the composition of the fusion partners. The method should produce cleaved product with authentic N- and C-termini, should not modify or otherwise adulterate the desired protein product, and should be tolerant to a wide range of conditions so that reaction components can be tailored to the physical characteristics of the fusion protein without seriously affecting the efficiency of the cleavage reaction. In addition, for biopharmaceutical production and applications, the cleaving reagent should not be from an animal source due to concerns about contamination by infectious agents.
An ideal choice for such a “universal” fusion protein cleaving method is use of the mammalian enzyme enterokinase (enteropeptidase). Enterokinase is the physiological activator of trypsinogen and cleaves with high specificity after the sequence (Asp
4
)-Lys (SEQ ID NO:34). Light et al, J. Protein Chem. 10:475-480(1991). It is possible to engineer the fusion protein to include a linker DNA sequence encoding the amino acid sequence recognized by enterokinase. See for example, Bollen et al., U.S. Pat. No. 4,828,988 (May 9, 1988); Rutter, U.S. Pat. No. 4,769,326 (Sep. 6, 1988); and Mayne et al., U.S.Pat. No. 4,745,069 (May 17, 1988). However, although extensive research efforts have been mounted by several different research groups since the first partial purification of bovine enterokinase more than 15 years ago, no one has yet been successful in cloning enterokinase. Porcine enterokinase was first isolated in the early 1970s (Maroux et al., J.Biol.Chem. 246:5031(1971))and bovine (Anderson et al., Biochemistry 16:3354(1977)) and human (Grant et al., Biochem. J. 155:243(1976)) enterokinases were isolated in the late 1970s. Liepnieks et al., J. Biol. Chem. 254:1677(1979) described an enterokinase having 35% carbohydrate, a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Subsequent studies of the light chain, i.e., the catalytic subunit, were reported in Light et al., J. Biol. Chem. 259:13195(1984). Most recently, Light et al., J. Protein Chem. 10:475(1991), disclosed what was later proven to be an incorrect partial amino-terminal sequence for the catalytic subunit of bovine enterokinase. To date, it has been impossible to obtain recombinantly produced enterokinase activity and there continues to exist a need for such a product.
BRIEF SUMMARY
The present invention provides novel purified nucleic acid sequences encoding enterokinase activity. Specifically provided is mammalian enterokinase activity, including human and bovine enterokinase and comprising the nucleic acid sequence as set forth in SEQ ID NO:1, encoding the catalytic light chain, as well as portions of the heavy chain. The sequence comprises 2581 nucleotides and includes the catalytic domain, i.e., nucleotides 1691 to 2398. A nucleotide sequence encoding this enterokinase activity and contained in the plasmid designated pEK-2/G1734 was deposited with the American Type Culture Collection (ATCC) on Feb. 2, 1993 and accorded the accession number 69232. In a further embodiment, the invention comprises the expression products of the novel sequences having enterokinase activity.
Nucleic acid forms such as genomic DNA (gDNA), complementary DNA (cDNA), and DNA prepared by de novo chemical synthesis from nucleotides, as well as DNA with deletions or mutations, allelic variants and sequences that hybridize thereto under stringent conditions (or which would hybridize but for the redundancy of the genetic code) are also within the contemplation of the invention so long as they encode polypeptides having enterokinase activity as defined below. Also, forms which contain modifications of the catalytic site of enterokinase which may allow for alteration of the specific cleavage site recognized by the enzyme are included. Further provided are novel messenger RNA (mRNA) sequences corresponding to these DNA sequences.
Association of nucleic acid sequences provided by the invention with homologous or heterologous species expression control sequences, such as promoters, operators, regulators, and the like, allows for in vivo and in vitro transcription to the corresponding mRNA which, in turn, allows translation of proteins and related poly- and oligo-peptides, in large quantities, having enterokinase activity. In a presently preferred expression system of the invention, enterokinase encoding sequences are operatively associated with a regulatory promoter sequence allowing for transcription and translation in a eukaryotic cell system to provide e.g., enterokinase polypeptides having protease activity. The novel nucleic acid sequences may optionally encode both the heavy chain and the light chain of enterokinase, or the light chain alone which surprisingly still provides enterokinase activity. The enterokinase activity of the invention may be generated from one or more expression vector(s) each comprising one or more portions of the enterokinase activity, or, alternatively, the enterokinase activity can be generated from one or more expression vector(s) contained in one or more cell lines, each of which express all or a portion of the enterokinase activity. Thus, the heavy and light chains may be separately expressed in separate cell lines if desired. In addition, the enterokinase activity can be produced as a fusion protein, e.g., using thioredoxin as the fusion partner. Optionally, the fusion partner can be all or part of yet another proteolytic enzyme, such as PACE, trypsinogen, and the like. Indeed, such an enterokinase fusion protein can contain an enterokinase cleavage site between the component protein domains, thereby allowing autocatalytic processing to separate the two domains and to yield mature, active enterokinase.
Incorporation of these sequences into prokaryotic and eukaryotic host cells by standard transformation and transfection processes, is also within the contemplation of the invention and is expected to provide useful enterokinase in quantities greatly in excess of those obtainable from tissue sources. The use of appropriate host cells provides for such post-translational modifications, e.g., truncation, glycosylation, etc., when needed to confer optimal biological activity on the expression products of the invention. Such appropriate host cells can include for example
E. coli,
CHO, yeast, and lepidoptera cells.
Novel protein products of the invention include those having the primary structural conformation (i.e., amino acid sequence) of
Duffy Patricia A.
Genetics Institute LLC
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