Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-11-12
2001-03-20
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S219000, C435S220000, C435S252330
Reexamination Certificate
active
06204008
ABSTRACT:
BACKGROUND OF THE INVENTION
The large scale production of compounds which are based, at least in part, on a dipeptide intermediate requires the ready availability of large quantities of the dipeptide intermediate at low cost. Dipeptides of virtually any amino acids may be synthetically prepared for this use, however, synthesis of the dipeptide can be time consuming and costly which reduces the efficiency of product production and increases production costs. These disadvantages are particularly important to very large scale production of the dipeptide intermediate-based compounds.
SUMMARY OF THE DISCLOSURE
A process for the production of dipeptide intermediate compounds is disclosed. These dipeptide intermediate compounds are produced by recombinant DNA techniques and are produced from a synthetic DNA sequence or from a naturally-occurring DNA sequence, both of which encode a protein or peptide having a specific repeating dipeptide sequence. The dipeptide intermediates are enzymatically liberated from the larger protein or peptide and further processed to provide the finished compound.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is drawn to a process for the preparation of dipeptide intermediate compounds. These intermediate compounds can be further processed to produce the finished compound. The dipeptide intermediates are produced in recombinant host cells through the expression of a recombinant DNA molecule, which encodes at least one dipeptide intermediate. The recombinant DNA molecule can be either a synthetic DNA molecule or a naturally occurring DNA sequence. The DNA encoding the dipeptide is cloned into an expression vector for expression in a recombinant host cell. Following expression in the recombinant host the dipeptide is purified for further processing. It may be necessary to separate the dipeptide from a larger polypeptide before it can be purified. A larger polypeptide containing the dipeptide intermediate may be comprised of multiple repeats of the dipeptide sequence, or the dipeptide may be present in one or more copies as part of a larger protein containing amino acid sequences that are not the dipeptide sequence. The repeats of the dipeptide sequence may be separated into individual dipeptides by enzymatic or chemical cleavage. The individual dipeptides may then be purified for further processing.
The recombinant host cells used to produce the dipeptide can be any host cell that is capable of expressing recombinant DNA molecules. It is readily apparent to those skilled in the art that virtually any recombinant host is suitable for use in the process of the present invention. The preferred host cells include, but are not limited, to bacteria and fungal cells including yeast. Bacterial and fungal host cells which are suitable for use in the present invention and are commercially available include, but are not limited, to
Sacccharomyces cerevisiae, Pichia pastoris, Escherichia coli, Aspergillus niger, Streptomyces lividans
, and
Bacillus subtilis.
It is also readily apparent to those skilled in the art that the expression vector for expressing the DNA encoding the dipeptide can be virtually any expression vector suitable for use in the chosen host cell. The preferred vectors include, but are not limited to, those suitable for use in bacteria and fungal cells including yeast. Bacterial and fungal expression vectors which are suitable for use in the present inventions and are commercially available include, but are not limited to those listed herein.
The separation of the dipeptide from the remaining non-dipeptide sequences, or the separation of individual dipeptides from dipeptide repeats is performed enzymatically using an enzyme which cleaves the polypeptide in the appropriate position without destroying the dipeptide intermediate. Enzymes suitable for use in the process of the present invention can be those which are commercially available or those produced by an organism which possesses the appropriate enzymatic activity. The selection of an appropriate enzyme will depend upon the particular dipeptide sequence being expressed.
The dipeptide may be purified either before or after it is separated from the remainder of the polypeptide or from repeats of the dipeptide. Standard chromatographic techniques are suitable for use in the process of the present invention to purify the individual dipeptides or the dipeptide-containing polypeptide. Such standard chromatographic techniques include, but are not limited to salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography, and hydrophobic interaction chromatography.
The cloned DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant peptide. Techniques for such manipulations are fully described in Sambrook, J., et al., supra, and are well known in the art.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and animal cells.
Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria-invertebrate cells. An appropriately constructed expression vector should contain; an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
A variety of mammalian expression vectors may be used to express recombinant peptide in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant peptide expression include, but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTI (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and &lgr;ZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to express recombinant peptide in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant peptide expression include, but are not limited to, pET11a (Novagen), lambda gt11 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express recombinant peptide in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant peptide expression include, but are not limited to, pYES2 (Invitrogen), Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to express recombinant peptide in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of peptide include, but are not limited to, pBlue Bac III (Invitrogen).
An expression vector containing DNA encoding the dipeptide intermediate may be used for expression of peptide in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic including, but not limited to, bacteria such as
E. coli
, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including, but not limi
Borneman W. Scott
Conder Michael J.
Goyal Anil
Vinci Victor A.
Fronda Christian L
Hand J. Mark
Merck & Co. , Inc.
Nashed Nashaat T.
Tribble Jack L.
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