Methods of RNA and protein synthesis
The present invention relates to methods for RNA and/or protein synthesis using in vitro or in vivo expression systems. More specifically, the present invention provides a method for RNA and/or protein synthesis characterized in that the concentration of alpha subunit of RNA polymerase, but not of other subunits, is increased in the cellular or cell-free system, comparing to its natural concentration existing in the cellular or cell-free system.
1. A method to increase RNA synthesis from a DNA template comprising:
a) providing a cell-free system comprising a bacterial cell-free extract;
b) adding a DNA template comprising a bacterial promoter with at least one UP element to said cell free system; and
c) recovering the synthesized RNA,
wherein the ratio of an α subunit of RNA polymerase to other subunits concentration in said cell-free system is increased as compared to the conventional ratio of two α, one β, one β′, and one σ.
2. The method according to claim 1 , wherein the bacterial promoter is an argC gene promoter of Bacillus stearothermophilus.
3. The method according to claim 2 , wherein the promoter comprises the sequence from nucleotide at position −89 to nucleotide at position +1 of the argC gene promoter of Bacillus stearothermophilus , when position +1 is the first nucleotide in mRNA of the argC gene.
4. The method according to claim 1 , wherein said cell-free system further comprises a purified thermostable RNA polymerase holoenzyme.
5. The method according to claim 4 , wherein said thermostable RNA polymerase holoenzyme is from Thermus thermophilus.
6. The method according to claim 1 , wherein the concentration of said α subunit of RNA polymerase is increased by adding a purified α subunit of RNA polymerase to the bacterial cell-free extract.
7. The method according to claim 6 , wherein said purified α subunit is added to a final concentration between 15 μg/ml and 200 μg/ml.
8. The method according to claim 6 , wherein said purified added α subunit of RNA polymerase is different from an α subunit present in the bacterial extract.
9. The method according to claim 8 , wherein said purified added α subunit is from E. coli, T. maritima or T. neapolitana.
10. The method according to claim 6 , wherein the purified added α subunit is purified from cells overexpressing a gene encoding an α subunit of RNA polymerase.
11. The method according to claim 1 , wherein the bacterial cell-free extract is prepared from cells overexpressing a gene encoding an α subunit of RNA polymerase.
12. The method according to claim 1 , wherein the UP element is a AT-rich region around 18–20 bp long.
13. The method according to claim 1 , wherein said bacterial cell-free extract is from E. coli cells.
14. The method according to claim 13 , wherein said E. coli cells are K12 A19 cells having a rna19 gdhA2 his-95 relA1l spoT1metB1 genotype.
15. A method to increase the production of a protein from a DNA template in a cell-free system comprising:
a) providing in a reaction mixture, a bacterial cell-free system;
b) adding to the reaction mixture the DNA template encoding a desired protein and a purified α subunit of a RNA-polymerase; and
c) recovering the produced protein,
wherein the DNA template comprises a bacterial promoter with at least one UP element.
16. The method according to claim 15 , wherein said purified α subunit is added to a final concentration between 15 μg/ml and 200 μg/ml.
17. The method according to claim 15 , wherein a DNA-binding regulatory protein is further added to the reaction mixture at step (b).
18. The method according to claim 15 , wherein said DNA template comprises an amplification product of an Open Reading Frame encoding the desired protein.
19. The method according to claim 18 , wherein said DNA template further comprises an additional DNA fragment, which is at least 3 bp long, located immediately downstream of the stop codon of said Open Reading Frame.
20. The method according to claim 18 , wherein said DNA template further comprises an additional DNA fragment containing a transcriptional terminator.
21. The method according to claim 20 , wherein said transcriptional terminator is a T7 phage transcriptional terminator.
22. The method according to claim 20 , wherein said additional DNA fragment is longer than 100 bp.
23. The method according to claim 20 , wherein said additional DNA fragment is longer than 200 bp.
24. The method according to claim 15 , wherein a thermostable RNA polymerase is further added in step b).
25. The method according to claim 24 , wherein said added RNA polymerase is a thermostable RNA polymerase from T. thermophilus.
26. The method according to claim 15 , wherein the bacterial promoter with at least one UP element is from the argC gene of Bacillus stearothermophilus.
27. The method according to claim 26 , wherein the bacterial promoter includes the sequence from nucleotide at position −89 to nucleotide at position +1 of the argC gene promoter of Bacillus stearothermophilus , when position +1 is the first nucleotide in mRNA of the argC gene.
28. A method for RNA or polypeptide synthesis from a DNA template comprising:
a) providing a bacterial cell-free extract;
b) adding a DNA template comprising a bacterial promoter with at least one UP element to said cell extract, and
c) recovering the synthesized RNA or polypeptide;
wherein the ratio of an α subunit of RNA polymerase to other subunits concentration in said cell-free extract is increased as compared to the conventional ratio of two α, one β, one β′, and one σ, by adding in said bacterial cell free extract a purified α subunit of RNA polymerase prepared from cells overexpressing a gene encoding said α subunit of RNA polymerase.