Cell lines for expressing enzyme useful in the preparation of amidated products
View Patent ↗Cell lines are provided for expressing peptidylglycine alpha-amidating monooxygenase (PAM), or one of its two catalytic domains. High levels of enzyme expression are achieved while utilizing a non-animal source, low protein tissue culture medium. A robust two-step downstream purification results in high enzyme purity. Resulting PAM, or its PHM catalytic domain, is used to catalyze the enzymatic conversion of X-Gly to X-alpha-hydroxy-Gly or X—NH 2 (X being a peptide or any chemical compound having a carbonyl group to which a glycine group can be covalently attached). Methods of preparing preferred cell lines are also set forth.
1. A CHO KI cell transfected with an expression vector for expressing peptidylglycine alpha-amidating monooxygenase, wherein said expression vector has a coding region encoding peptidylglycine alpha-amidating monooxygenase, wherein the coding region is operably linked to a control region including a ribosome binding site, a promoter, and an SV40 enhancer upstream of said promoter,
wherein the promoter is an inducible human metallothionein IIa promoter and is located immediately upstream of a start site of the coding region,
wherein the SV40 enhancer is located immediately upstream of the inducible human metallothionein IIa promoter,
wherein the peptidylglycine alpha-amidating monooxygenase consists of a signal peptide at amino acid residues 1 to 25, a pro-region at amino acid residues 26 to 41, and a mature peptidylglycine alpha-amidating monooxygenase at amino acid residues 42 to 715 as set forth in SEQ ID NO: 2,
wherein the CHO K1 cell is capable of expressing a soluble peptidylglycine alpha-amidating monooxygenase.
2. A recombinant expression vector having a coding region with encoding peptidylglycine alpha-amidating monooxygenase, wherein the coding region is operably linked to a control region including a ribosome binding site, a promoter and an SV40 enhancer upstream of said promoter,
wherein the promoter is an inducible human metallothionein IIa promoter and is located immediately upstream of a start site of the coding region,
wherein the SV40 enhancer is located immediately upstream of the inducible human metallothionein IIa promoter,
wherein the peptidylglycine alpha-amidating monooxygenase consists of a signal peptide at amino acid residues 1 to 25, a pro-region at amino acid residues 26 to 41, and a mature peptidylglycine alpha-amidating monooxygenase at amino acid residues 42 to 715 as set forth in SEQ ID NO: 2,
wherein the recombinant expression vector is capable of expressing a soluble peptidylglycine alpha-amidating monooxygenase.
3. A method of preparing a cell line for the expression of peptidylglycine alpha-amidating monooxygenase, said method comprising the steps of:
(A) Transfecting potential host cells in the presence of first, second and third expression vectors, wherein said first vector includes a coding region encoding a first selectable marker, wherein said second vector includes a coding region encoding a second selectable marker, wherein said third vector includes a coding region encoding peptidylglycine alpha-amidating monooxygenase, wherein the coding region is operably linked to a control region including a ribosome binding site, a promoter and an SV40 enhancer upstream of said promoter
wherein the SV40 enhancer is located immediately upstream of the inducible human metallothionein IIa promoter,
wherein the peptidylglycine alpha-amidating monooxygenase consists of a signal peptide at amino acid residues 1 to 25, a pro-region at amino acid residues 26 to 41, and a mature peptidylglycine alpha-amidating monooxygenase at amino acid residues 42 to 715 as set forth in SEQ ID NO: 2,
wherein the third vector is capable of expressing a soluble peptidylglycine alpha-amidating monooxygenase,
wherein the concentration ratio of said third vector to said first vector is at least 3:1, and wherein the concentration ratio of said third vector to said second vector is at least 3:1;
(B) Subjecting the cells resulting from step (A) to selectable pressure to select cells that have been transfected with said first vector;
(C) Subjecting the cells resulting from step (B) to selectable pressure to select cells that have been transfected with said second vector;
(D) Subjecting the cells resulting from step (C) to limiting dilution and selecting cells expressing soluble peptidylglycine alpha-amidating monooxygenase.
4. The method of claim 3 further comprising the step of amplifying peptidylglycine alpha-amidating monooxygenase by subjecting cells resulting from step (D) to amplifying selective pressure.
5. The method of claim 3 wherein said first selectable marker is a gene encoding neomycin resistance.
6. The method of claim 3 wherein said second selectable marker is a gene encoding dihydrofolate reductase.
7. The method of claim 4 wherein at least one of said selectable markers is a gene encoding dihydrofolate reductase and wherein said amplification is conducted in the presence of methotrexate.
8. The method of claim 3 wherein the concentration ratio of said third vector to said first vector is at least 10:1 and the concentration ratio of said third vector to said second vector is at least 10:1.
9. The method of claim 3 wherein said third vector includes a coding region having nucleic acids encoding peptidylglycine alpha-amidating monooxygenase, operably linked to a control region including a ribosome binding site, a promoter and an SV40 enhancer upstream of said promoter.
10. The method of claim 3 wherein said third vector includes a coding region having nucleic acids encoding peptidylglycine alpha-amidating monooxygenase, operably linked to a control region including a ribosome binding site, human metallothionein to a promoter and an enhancer upstream of said promoter.
11. The method of claim 10 wherein said enhancer is an SV40 enhancer.
12. The method of claim 3 wherein said potential host cells include CHO K1 cells.
13. A cell line that results from the method of claim 3 .
14. A cell line comprising host cells transfected with the vector of claim 2 .
15. The cell line deposited as ATCC accession number PTA 6784.