Expression and purification of Cas enzymes
Described herein are methods for the expression and purification of Cas13a and methods for detecting target RNA using Cas13a.
1. A method for expressing and purifying a Cas13a protein, the method comprising:
(a) inserting a nucleotide sequence encoding a polypeptide having the polypeptide sequences of SEQ ID NO: 10 into an expression plasmid;
(b) transforming one or more cells with the expression plasmid;
(c) inducing expression of the transformed plasmid;
(d) isolating the cells;
(e) extracting the Cas13a protein; and
(f) purifying the protein using affinity purification and ion exchange purification.
2. The method of claim 1 , wherein the cell comprises E. coli BL21(DE3).
3. The method of claim 1 , wherein the expression plasmid comprises pET28 or pET28-MBP-TEV plasmids.
4. The method of claim 1 , wherein the nucleotide sequence is inserted into the expression plasmid using isothermal assembly.
5. The method of claim 1 , wherein the affinity purification comprises a nickel or a maltose affinity media.
6. The method of claim 1 , wherein the affinity purification comprises affinity chromatography comprising:
(f(1) a equilibrating a nickel affinity column with a binding buffer and loading the extracted Cas13a protein;
f(2) washing the nickel affinity column with a wash buffer; and
f(3) eluting the affinity purified Cas13a protein from the nickel affinity column using elution buffer.
7. The method of claim 1 , wherein the affinity purification comprises affinity chromatography comprising:
f(1) equilibrating a maltose affinity column with a binding buffer and loading the extracted Cas13a protein;
f(2) washing the maltose affinity column with a wash buffer; and
f(3) eluting the affinity purified Cas13a protein from the maltose affinity column using elution buffer.
8. The method of claim 1 , wherein the ion exchange purification comprises a cation exchange media.
9. The method of claim 1 , wherein the ion exchange purification comprises cation exchange chromatography comprising:
(1) equilibrating a cation exchange column with a binding buffer and loading the extracted Cas13a protein;
(2) washing the cation exchange column with a wash buffer; and
(3) eluting the cation exchange purified Cas13a protein from the cation exchange column using an elution buffer.
10. The method of claim 1 , further comprising concentrating the purified Cas13a protein to approximately 10 mg/mL.
11. The method of claim 10 , further comprising dialyzing the concentrated purified Cas13a protein.
12. A method for purifying a recombinant Cas13a protein, the method comprising:
(a) providing an expressed recombinant Cas13a protein having the polypeptide sequence of SEQ ID NO: 10;
(b) performing an affinity purification comprising a nickel affinity media or a maltose affinity media;
(c) performing an ion exchange purification comprising a cation exchange media; and
(d) collecting the purified Cas13a protein.
13. The method of claim 12 , further comprising concentrating the purified Cas13a protein to approximately 10 mg/mL.
14. The method of claim 13 , further comprising dialyzing the concentrated purified Cas13a protein against three rounds of dialysis buffer.