Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-04-02
2001-12-18
Prouty, Rebecca (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S183000, C435S195000, C435S200000, C435S252300, C435S320100, C435S252330, C536S023200, C424S093200
Reexamination Certificate
active
06331425
ABSTRACT:
PRIOR FOREIGN APPLICATIONS
This application is a 35 USC §371 filing of PCT/GB96/01577, filed Jul. 1, 1996 and claims priority from GB Patent Application Number 9513683.4, filed July 5, 1995.
This invention relates to a recombinant protein having bacteriophage endosialidase activity, to a process for the production thereof and to recombinant expression systems for use in the production thereof.
Bacteriophage E is a member of the PK1A-PK1E family of phages; these phages were isolated originally from European sewage to aid in the clinical identification of
Escherichia coli
K1 infections, which can result in high mortality rates in cases of neonatal meningitis. Bacteriophage E endosialidase (K I E endosialidase) is thought to be the enzyme responsible for initial binding to host bacteria by specifically recognising and hydrolysing the &agr;-2,8-linked poly-N-acetylneuraminic acid (polysialic acid/PSA) carbohydrate polymers of the K1 glycocalyx. &agr;-2,8-linked PSA is also expressed on the cell surface of several other pathogenic bacteria, and various tumour cells and cell lines. It has been proposed in U.S. Pat. No. 4,695,541 that K1E endosialidase could be used in the diagnosis and therapy of K1 meningitis, septicaemia or bacteraemia due to the enzyme's high specificity for hydrolysing &agr;-2,8-sialosyl linkages. PSA has been suggested as an oncodevelopmental marker in human tumours of the kidney and neuroendocrine tissues and also may contribute to the invasive and metastatic potential of some tumours.
In J. Bacteriol., 1993, 175, 4354-4363, there are described attempts to obtain enzymatically active protein by expression from a DNA construct derived from the related KIF phage; these attempts were unsuccessful.
It has now been found that protein having bacteriophage endosialidase enzymatic activity, i.e. a protein which specifically binds to or cleaves &agr;-2,8-polysialic acid, can be obtained by expression from a DNA construct which is derivable from the KIE endosialidase gene and is cloned into an expression vector which expresses a polypeptide which adds to the N-terminus of the endosialidase sequence.
Accordingly, the present invention provides, in one aspect, a recombinant protein having bacteriophage endosialidase enzymatic activity obtainable by expression from a recombinant vector comprising a DNA sequence encoding a bacteriophage endosialidase linked to a DNA sequence of an expression vector which expresses a polypeptide which adds to the N-terminus of the endosialidase, or an analogue of said protein which is a mutant, functional fragment or derivative of said protein having endosialidase enzymatic activity.
The mutant may be, for example, a protein having an amino acid substituted or deleted at one or more positions- The functional fragment may be C- or N-terminal shortened fragment or a fragment from within the polypeptide chain which has endosialidase enzymatic activity. The derivative may be, for example, a pharmaceutically acceptable salt with an acid such as hydrochloric acid, sulphuric acid, phosphoric acid, pyrophosphoric acid, benzenesulphonic acid, p-toluenesulphonic acid, methanesulphonic acid, lactic acid, palmic acid, tartaric acid, ascorbic acid, or citric acid; with a base, usually a nitrogen containing base such as sodium, potassium, magnesium or ammonium nitrogen-containing base; or an internal salt.
In another aspect, the present invention provides a recombinant vector comprising a DNA sequence encoding a bacteriophage endosialidase linked to a DNA sequence of an expression vector which expresses a polypeptide which adds to the N-terminus of the endosialidase, said recombinant vector being capable of directing expression of said protein in a compatible host cell.
In a further aspect the present invention provides a process for the production of a protein having bacteriophage E endosialidase enzymatic activity which comprises culturing a host cell transformed with a recombinant vector as hereinbefore defined under conditions allowing expression of said protein and isolating the protein thereby produced. In a yet further aspect, the present invention provides a host cell transformed with a recombinant vector as hereinbefore defined.
Preferred protein according to the invention is a protein obtainable by expression from a recombinant vector as hereinbefore defined in which the DNA sequence encoding the endosialidase is derived from a DNA construct encoding amino acid residues encoded by nucleotides 172 to 1744 of the bacteriophage E endosialidase gene, i.e. nucleotides 172 to 1744 of SEQ ID No. 1 as hereinafter defined, or a mutant, functional fragment or derivative of said protein which has endosialidase enzymatic activity. An especially preferred protein according to the invention is a protein obtainable by expression from a recombinant vector as hereinbefore defined in which the DNA sequence encoding the endosialidase is derived from a DNA construct encoding amino acid residues encoded by nucleotides 1 to 2436 of the bacteriophage E endosialidase gene, i.e. nucleotides 1 to 2436 of SEQ. ID NO. 1 as hereinafter defined, or a mutant, functional fragment or derivative of said protein having endosialidase enzymatic activity
The protein of the invention is generally expressed in the form of a fusion protein comprising the endosialidase linked, directly or through a spacer, to a polypeptide derived from the expression vector, i.e. the vector used for expression of the protein in a suitable host cell, a preferred such polypeptide being glutathione S-transferase. Where it is desired that the polypeptide components of the fusion protein should be separable, if the fusion protein does not naturally contain a region which can be specifically cleaved chemically or enzymatically, such a region can be inserted using conventional procedures. Examples of selective cleaving reagents or cleaving enzymes for fusion proteins are V8 protease, trypsin, thrombin, factor X, CNBr, peptidase ysc&agr; and yscF.
In a particularly preferred embodiment of the invention, the protein of the invention is in the form of a fusion protein comprising bacteriophage E endosialidase linked to glutathione S-transferase, the fusion protein preferably having a molecular weight of about 100 kDa.
A DNA construct, i.e. recombinant DNA molecule, suitable for the expression of a protein according to the invention may be an isolated DNA fragment encoding a bacteriophage endosialidase, for example consisting only of the coding region or prolonged by homologous or heterologous DNA sequences. The construct may be a DNA fragment encoding the endosialidase cloned into a suitable cloning vector, preferably a bacterial vector such as pBR317, pBR322, pUC18, pSF2124 or, especially, Bluescript SK
+
. Where such a clone lacks convenient restriction sites with which to isolate solely the endosialidase open reading frame, it may be amplified by a polymerase chain reaction (PCR) using primers incorporating the restriction sites required.
The DNA fragment encoding the bacteriophage endosialidase may be obtained from genomic bacteriophage E DNA or a synthetic DNA that is substantially homologous thereto, i.e. is 80-100% homologous thereto. Bacteriophage E can be purified and total genomic DNA can be extracted using conventional procedures. The extracted DNA can then be digested with an appropriate restriction enzyme such as Bgl II, Eco RI, Hinc II, Hind III, Bam HI or Pst I. The digestion product can be subjected to preparative electrophoresis with low-melting point agarose gel to enrich DNA fractions of a certain length in order to enrich DNA fragments encoding the protein of the invention.
When a nucleotide sequence encoding the bacteriophage endosialidase, or an amino acid sequence thereof, is known, DNA encoding the endosialidase can also be prepared by methods leading directly to the desired DNA such as conventional PCR procedures or in vitro chemical synthesis.
For expression of a protein of the invention, the DNA construct is cloned into an expression vector which expresses a polypeptide whic
Bryant Jonathan Mark
Luzio John Paul
Taylor Peter William
Endozyme Limited
Heslin Rothenberg Farley & Miesit P.C.
Prouty Rebecca
Rao Manjunath N.
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