Claims
- 1. A method for a large-scale production of antigen-specific intact antibody, said method comprising steps:
(a) isolating cDNA, mRNA or genomic DNA of genes for antibody light and heavy chains and assembling the antibody genes into expression cassettes containing the cDNA; (b) preparing a recombinant P. pastoris yeast expression vector; (c) constructing a recombinant P. pastoris yeast expression plasmid containing the expression cassettes of cDNA of the light and heavy chain genes encoding the antibody; (d) cloning the antibody expression cassettes into the P. pastoris expression vector to generate recombinant plasmid; (e) transforming Saccharomyces cerevisiae with the recombinant plasmid by placing said expression cassettes under the control of the AOX1 promoter fused to a Saccharomyces cerevisiae α-mating factor signal sequence; (f) amplifying and isolating the recombinant plasmid; (g) preparing and transforming P. pastoris with BglII, NotI, SacI, SalI or Stul-linearized recombinant plasmid replacing the yeast chromosomal AOX1 sequence with AOX1-antibody gene cassettes of the recombinant plasmid; (h) selectively growing the recombinants; (i) screening yeast transformation colonies for a recombinant antibody expression; (j) analyzing putative positive yeast clones for chromosomal integrates of the expression cassettes of heavy and light chain cDNAs; (k) confirming the integrity of the DNA insert or junction sequence; (1) inducing the recombinant antibody expression; (m) confirming the intactness of the expression cassettes inserts with PCR and Northern blot analysis; (n) detecting the presence of the recombinant antibody by Western blot; and (o) testing the recombinant antibody for specific antigen-antibody binding.
- 2. The method of claim 1 wherein the antibody genes are assembled into the expression cassettes by subcloning the antibody light and heavy chain cDNA in tandem EcoRI-BglII/BsmBI fragments flanked by a P. pastoris signal sequence, preceded by a P. pastoris promoter at the 5′ terminus and a P. pastoris yeast transcription termination sequence at the 3′-terminus.
- 3. The method of claim 2 wherein the signal sequence is α-factor and wherein the promoter is AOX1-P.
- 4. The method of claim 3 wherein the yeast expression vector is pPICZα.
- 5. The method of claim 4 wherein the yeast expression vector is prepared by restriction digestion with EcoRI and BamHI.
- 6. The method of claim 5 wherein the recombinant plasmid is pPICZαLH.
- 7. The method of claim 6 wherein the recombinant expression plasmid pPICZαLH is constructed by cloning the antibody genes expression cassettes into the P. pastoris expression vector.
- 8. The method of claim 7 wherein the replacement of the yeast chromosomal AOX1 with AOX1-antibody gene cassettes is by homologous recombination replacement.
- 9. The method of claim 8 wherein the selective growth of the recombinants is performed on a medium containing zeocin.
- 10. The method of claim 9 wherein the selective growth of the recombinants is performed on a medium containing g418, trimethoprin, or a compound that limits the growth of wild type P. pastoris.
- 11. The method of claim 10 wherein the screening of transformed colonies is by colony-immunoblotting.
- 12. The method of claim 11 wherein the screening is by PCR or by restriction analysis.
- 13. The method of claim 12 wherein the integrity of the DNA inserts or junction sequence is confirmed by nucleotide sequence analysis.
- 14. Intact antigen-specific antibodies produced by P. pastoris transformed with mouse, humanized mouse or human immunoglobulin genes, said antibody produced by the process comprising steps:
(a) isolating cDNA, mRNA or genomic DNA of genes for antibody light and heavy chains and assembling the antibody genes into expression cassettes containing the cDNA; (b) preparing a recombinant P. pastoris yeast expression vector; (c) constructing a recombinant P. pastoris yeast expression plasmid containing the expression cassettes of cDNA of the light and heavy chain genes encoding the antibody; (d) cloning the antibody expression cassettes into the P. pastoris expression vector to generate recombinant plasmid; (e) transforming Saccharomyces cerevisiae with the recombinant plasmid by placing said expression cassettes under the control of the AOX1 promoter fused to a Saccharomyces cerevisiae α-mating factor signal sequence; (f) amplifying and isolating the recombinant plasmid; (g) preparing and transforming P. pastoris with BglII, NotI, SacI, SalI or Stul-linearized recombinant plasmid replacing the yeast chromosomal AOX1 sequence with AOX1-antibody gene cassettes of the recombinant plasmid; (h) selectively growing the recombinants; (i) screening yeast transformation colonies for a recombinant antibody expression; (j) analyzing putative positive yeast clones for chromosomal integrates of the expression cassettes of heavy and light chain cDNAs; (k) confirming the integrity of the DNA insert or junction sequence; (1) inducing the recombinant antibody expression; (m) confirming the intactness of the expression cassettes inserts with PCR and Northern blot analysis; (n) detecting the presence of the recombinant antibody by Western blot; and (o) testing the recombinant antibody for specific antigen-antibody binding and intactness.
- 15. The antibody of claim 14 wherein the antibody genes are assembled into the expression cassettes by subcloning the antibody light and heavy chain cDNA in tandem EcoRI-BglII/BsmBI fragments flanked by a P. pastoris signal sequence, preceded by a P. pastoris promoter at the 5′terminus and a P. pastoris yeast transcription termination sequence at the 3′-terminus.
- 16. The antibody of claim 15 produced by P. pastoris transformed with human immunoglobulin genes.
- 17. The antibody of claim 15 produced by P. pastoris transformed with humanized mouse immunoglobulin genes.
- 18. The antibody of claim 15 produced by P. pastoris transformed with mammalian or mouse immunoglobulin genes.
- 19. A recombinant P. pastoris yeast expression vector containing dual expression cassettes, each carrying a cDNA copy of immunoglobulin light and heavy chain.
- 20. An expression system comprising P. pastoris transformed with antibody genes for production of a recombinant antigen-specific intact antibody.
- 21. P. pastoris yeast transformed with expression cassettes carrying a cDNA copy of immunoglobulin heavy and light chain suitable for large-scale production of intact antibodies.
Parent Case Info
[0001] This application is based on and claims priority of the Provisional Application Ser. No. 60/105,259 filed on Oct. 22, 1998.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60105259 |
Oct 1998 |
US |