Compositions and methods of using a synthetic Dnase I
View Patent ↗Compositions and method for making and using a synthetic bovine DNase I are disclosed. More particularly, the sbDNase I of the present invention is a versatile enzyme that cleaves DNA nonspecifically to release 5′-phosphorylated nucleotides. The sbDNase I molecules of the present invention find particular use in a wide range of molecular biology applications, including: degradation of contaminating DNA after RNA isolation; RNA clean-up prior to, or in conjunction with, RT-PCR after in vitro transcription; identification of protein binding sequences on DNA (DNase I footprinting); prevention of clumping when handling cultured cells; tissue dissociation and creation of fragmented DNA for in vitro recombination reactions.
1. An isolated and purified nucleic acid comprising a nucleic acid encoding the protein of SEQ ID NO: 2 for a DNase I.
2. The nucleic acid of claim 1 , further comprising a synthetic nucleic acid sequence optimized for microbial expression.
3. The nucleic acid of claim 1 , wherein the DNase I comprises a synthetic bovine DNase I further comprising an E13R mutation.
4. The nucleic acid of claim 1 , wherein the DNase I comprises a synthetic bovine DNase I further comprising an N74K mutation.
5. The nucleic acid of claim 1 , wherein the DNase I comprises a synthetic bovine DNase I further comprising an E13R mutation and an N74K mutation.
6. The nucleic acid of claim 1 , wherein the nucleic acid further comprises a nucleic acid segment encoding a leader sequence.
7. The nucleic acid of claim 1 , wherein the nucleic acid comprises a nucleic acid segment encoding an alpha mating factor leader sequence.
8. The nucleic acid of claim 1 , wherein the nucleic acid comprises a codon-optimized bovine DNase I nucleic acid encoding SEQ ID NO: 2.
9. The nucleic acid of claim 1 , wherein the protein encoded by the nucleic acid comprises an about 95 percent identity of higher with a codon-optimized bovine DNase I of SEQ ID NO: 2.
10. An expression vector comprising a nucleic acid encoding the protein of SEQ ID NO: 2 for a DNase I operably linked to a promoter recognized by a host cell transformed with the vector.
11. The expression vector of claim 10 , wherein the host cell is a yeast cell.
12. The expression vector of claim 10 , wherein the host cell comprises Pichia pastoris.
13. An isolated and purified nucleic acid comprising a nucleic acid sequence encoding a protein of at least about a 95% identity with a nucleic acid sequence encoding the protein of SEQ ID NO: 2 for a DNase I.
14. A host cell transformed with an expression vector comprising a nucleic acid encoding an amino acid sequence of SEQ ID NO: 2 for a synthetic bovine DNase I.
15. The host cell of claim 14 , wherein the host cell comprises a yeast cell.
16. The host cell of claim 14 , wherein the host cell comprises Pichia pastoris.
17. A process for making a protein with DNase activity comprising the steps of:
transforming a host cell with an isolated nucleic acid comprising a nucleotide sequence encoding a DNase Protein with at least about an 95% identity with SEQ ID NO: 2 for a DNase; and
culturing the host cell under conditions such that the DNase protein is produced by the host cell.
18. A synthetic bovine DNase I produced by a process comprising:
culturing a yeast host cell transformed with an expression vector comprising a DNA sequence comprising the nucleotide sequence encoding the synthetic bovine DNase I as shown in FIG. 1 (SEQ ID NO:2),
expressing the synthetic bovine DNase I in the cultured yeast host cell; and
isolating the synthetic bovine DNase I.
19. A process for making a synthetic bovine DNase I comprising the steps of:
transforming a host cell with a nucleic acid molecule that encodes the bovine DNase I comprising an amino acid sequence of SEQ ID NO: 2; and
culturing the host cell under conditions in which the bovine DNase I is produced by the host cell.
20. The process of claim 19 , wherein the host cell comprises Pichia pastoris.
21. The process of claim 19 , wherein the host cell produces at least 1.0 mg/L bovine DNase I protein.
22. The process of claim 19 , wherein the host cell produces at least 7.5 mg/L bovine DNase I protein.
23. A synthetic bovine DNase I made by the process of claim 19 .