Cell-free protein expression using rolling circle amplification product
Methods for in vitro transcription and translation from an RCA product are provided. The methods comprise providing a double-stranded RCA product, wherein the double-stranded RCA product consists essentially of tandem repeats of a minimalistic expression sequence. The methods further comprise expressing a protein from the double-stranded RCA product in a cell-free expression system.
1. A method for in vitro transcription and translation, comprising:
providing a double-stranded rolling circle amplification (RCA) product, wherein the double-stranded RCA product consists essentially of tandem repeats of a minimalistic expression sequence; and
expressing a protein from the double-stranded RCA product in a cell-free expression system,
wherein the minimalistic expression sequence consists essentially of a promoter, an open reading frame, a ribosomal binding site and a translational termination sequence.
2. The method of claim 1 , wherein the minimalistic expression sequence is devoid of any extraneous sequences that are required for propagation of a plasmid in a host cell.
3. The method of claim 2 , wherein the double-stranded RCA product is provided to the cell-free expression system without any further processing.
4. The method of claim 1 , wherein the double-stranded RCA product comprises a thioated nucleotide.
5. The method of claim 1 , wherein the minimalistic expression sequence further consists essentially of a transcriptional termination sequence, an insulator sequence, or a combination thereof.
6. The method of claim 1 , wherein the open reading frame comprises a codon-optimized sequence for enhancing rate of translation.
7. The method of claim 1 , wherein the open reading frame comprises a tag sequence for purification of the protein.
8. The method of claim 1 , wherein the cell-free expression system comprises a prokaryotic cell extract, a eukaryotic cell extract, or a combination thereof.
9. A method for in vitro transcription and translation, comprising:
providing a deoxyribonucleic acid (DNA) mini-circle, wherein the DNA mini-circle consists essentially of a minimalistic expression sequence;
generating a double-stranded rolling circle amplification (RCA) product via rolling circle amplification of the DNA minicircle; and
expressing a protein from the double-stranded RCA product in a cell-free expression system,
wherein the minimalistic expression sequence consists essentially of a promoter, an open reading frame, a ribosomal binding site and a translational termination sequence.
10. The method of claim 9 , wherein the minimalistic expression sequence is devoid of any extraneous sequences that are required for propagation of a plasmid in a host cell.
11. The method of claim 10 , wherein the double-stranded RCA product is provided to the cell-free expression system without any further processing.
12. The method of claim 9 , wherein the double-stranded RCA product comprises a thioated nucleotide.
13. The method of claim 9 , wherein the minimalistic expression sequence further consists essentially of an insulator sequence, a transcriptional termination sequence, or a combination thereof.
14. The method of claim 9 , wherein the open reading frame comprises a tag sequence for purification of the expressed protein.
15. The method of claim 9 , wherein the rolling circle amplification is performed using a final concentration of deoxyribonucleoside triphosphates (dNTPs) in a range of about 10 μM to about 10 mM.
16. The method of claim 9 , wherein the rolling circle amplification is performed using a random primer mixture comprising a nucleotide analogue.
17. The method of claim 16 , wherein the nucleotide analogue is an inosine, a Locked Nucleic Acid (LNA) nucleotide, a Peptide Nucleic Acid (PNA) nucleotide, a thioated nucleotide, 2-amino-deoxyadenosine, 2-thio-deoxythymidine, a polycation nucleotide, or a Zip Nucleic Acid (ZNA) polycation modified nucleotide.
18. The method of claim 16 , wherein the random primer mixture has a sequence +N+N(atN)(atN)(atN)*N.
19. The method of claim 9 , wherein the cell-free expression system comprises a prokaryotic cell extract, a eukaryotic cell extract, or a combination thereof.
20. The method of claim 9 , wherein the open reading frame comprises a codon-optimized sequence for enhancing rate of translation.