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Last Updated: March 29, 2024

Claims for Patent: 10,227,595


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Summary for Patent: 10,227,595
Title:Universal protein overexpression tag comprising ramp function, and application thereof
Abstract: Provided is a ramp tag capable of solving instability in translation rate resulting from poor compatibility between codons in a foreign gene and a host when expressing a recombinant protein in E. coli. Unlike the conventional codon optimization or codon deoptimization method for solving the problem of rare codons, the present invention increases an expression efficiency of a target protein by merely having the ramp tag be fused with a target gene or independently expressed, without changing the original codon sequence, thereby allowing tRNA to be reused. Thus, the present invention provides a novel method for increasing recombinant protein expression which is capable of reducing costs and time in comparison to the codon optimization method that artificially synthesizes DNA sequences. Therefore, it is expected that the method of the present invention will be able to be used in production of high value-added pharmaceuticals or industrial enzymes.
Inventor(s): Kim; Geun-Joong (Gwanju, KR), Park; Won Ji (Gwangju, KR), You; Sung-Hwan (Gwangju, KR), Lee; Jin-Young (Gwangju, KR), Lee; Eun-Bin (Gwangju, KR), Min; Sa-Young (Gwangju, KR)
Assignee: INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY (Gwangju, KR)
Application Number:14/773,529
Patent Claims:1. A method of producing a nucleic acid expression vector comprising a first nucleic acid sequence encoding a target protein (target gene sequence) and a second nucleic acid sequence encoding a ramp tag sequence, said method comprising: (a) identifying rare codons of a host cell by determining a frequency of usage of codons by the host cell and a number of isoacceptor tRNA of codons of the host cell, wherein the rare codons have the frequency of usage of the codons of 0.5 to 3% and the number of isoacceptor tRNA of 0 to 2; (b) determining which of the rare codons identified in (a) is appeared in the target gene sequence; (c) identifying a preferred codon which appears in the target gene sequence with a frequency of greater than 2%; (d) providing the ramp tag sequence comprising more than one rare codons and containing the preferred codon between the individual rare codons; and (e) producing the nucleic acid expression vector comprising the target gene sequence and the second nucleic acid sequence, wherein the ramp tag sequence contains 1-20 codons.

2. The method of claim 1, wherein (b) comprises determining a frequency and a position of the rare codons of (a) in the target gene sequence, and wherein the ramp tag sequence has a rare codon of the lowest frequency in the target gene sequence, at its 5'-end.

3. The method of claim 1, wherein the host cell is selected from the group consisting of E. coli, yeast, a Chinese hamster ovary cell, a human cell, and a plant cell.

4. The method of claim 3, wherein the host cell is selected from the group consisting of E. coli K-12, Saccharomyces cerevisiae, CHO-K1, HEK 293t, and 326-GFP.

5. The method of claim 1, wherein the target gene sequence encodes a peptide selected from the group consisting of an esterase, .beta.-glucosidase, cytolysin A (ClyA), recombinant antibody (single chain Fv: scFv), asparaginase B, tetra-cell adhesion molecule (T-CAM), B3(Fv)PE38, chloramphenicol acetyltransferase (CAT), neopullulanase, interleukin-1, interleukin-32, anti-B-lymphocyte antigen CD20 (anti-CD20), and anti-tumor necrosis factor alpha (anti-TNF.alpha.).

6. An expression vector to express an exogenous target protein in a host cell, said expression vector comprises a first nucleic acid sequence encoding the exogenous target protein (an exogenous target gene sequence) and a second nucleic acid sequence encoding a ramp tag sequence, wherein the ramp tag sequence comprises a rare codon of the host cell, said rare codon having a frequency of codon usage of 0.5 to 2% and a number of isoacceptor tRNA of 0 to 2, and said rare codon being also present in the exogenous target gene sequence, wherein the ramp tag sequence is selected from the group consisting of SEQ ID NOS: 1 to 3, 5, 6, and 11 to 45.

7. A method of increasing an expression efficiency of an exogenous target protein in a host cell, comprising: transforming the host cell with the expression vector of claim 6; and culturing the transformed host cell in a culture medium that is suitable for expressing the exogenous target protein.

8. The method of claim 7, wherein the ramp tag sequence of the expression vector comprises more than one rare codons and contains a preferred codon between the individual rare codons, said preferred codon being a codon which appears in the exogenous target gene with a frequency of greater than 2%.

9. The method of claim 7, wherein the host cell is selected from the group consisting of E. coli K-12, Saccharomyces cerevisiae, CHO-K1, HEK 293t, and 326-GFP.

10. The method of claim 7, wherein the exogenous target protein is selected from the group consisting of esterase, .beta.-glucosidase, cytolysin A, single chain Fv: scFv, asparaginase B, tetra-cell adhesion molecule, B3(Fv)PE38, chloramphenicol acetyltransferase, neopullulanase, interleukin-1, interleukin-32, anti-B-lymphocyte antigen CD20, and anti-tumor necrosis factor alpha.

11. The method of claim 7, wherein the ramp tag sequence is positioned so as to be fused to the 5' end or the 3' end of the exogenous target gene sequence, or positioned apart from the target gene so as to be independently translated from the exogenous target gene.

12. The method of claim 7, wherein the expression vector further comprises an additional tag selected from the group consisting of His tag, T7 tag, S-tag, Flag-tag, HA-tag, V5 epitope, PelB, and Xpress epitope.

13. The method of claim 12, wherein the expression vector further comprises a third nucleic acid sequence encoding an additional protein which forms a fusion protein with the exogenous target protein, said addition protein being selected from the group consisting of glutathione S-transferase (GST), maltose binding protein (MBP), transcription termination and antitermination (NusA), CREB binding protein (CBP), green fluorescent protein (GFP), thioredoxin, mistic, sumo and Disulfide-bond isomerase (DSB).

14. A method of separating a target protein from a culture solution containing the target protein and impurities, comprising: allowing the culture solution pass through an affinity chromatography so as the target protein to bind to the affinity chromatography; and eluting the target protein from the affinity chromatography, wherein the target protein is expressed from the expression vector of claim 6, wherein the ramp tag sequence of the expression vector is coupled to a histidine rare codon affinity chromatography.

15. The method of claim 14, wherein the affinity chromatography employs Ni-NTA resin to which a histidine residue binds, said histidine residue being encoded by the histidine rare codon.

16. The expression vector of claim 6, which further comprises a protein cleavage site-encoding sequence, said protein cleavage site-encoding sequence being placed between the ramp tag sequence and the exogenous target gene sequence.

17. The expression vector of claim 16, wherein the protein cleavage site is a peptide sequence recognized by an enzyme selected from the group consisting of lgA-protease, granzyme B, Tev protease, prescission protease, thrombin, a factor Xa, or enterokinase.

18. A host cell comprising the expression vector of claim 6.

19. The expression vector of claim 6, wherein the ramp tag sequence has a rare codon of the lowest frequency of codon usage at its 5'-end.

20. The expression vector of claim 6, wherein the ramp tag sequence comprises more than one rare codons and contains a preferred codon between the rare codons, said preferred codon being a codon which appears in the exogenous target gene with a frequency of greater than 2%.

21. The host cell of claim 18, which is selected from the group consisting of E. coli, yeast, a Chinese hamster ovary cell, a human cell, and a plant cell.

22. The host cell of claim 21, which is selected from the group consisting of E. coli K-12, Saccharomyces cerevisiae, CHO-K1, HEK 293t, and 326-GFP.

23. The expression vector of claim 6, wherein the exogenous target gene encodes a peptide selected from the group consisting of an esterase, (3-glucosidase, cytolysin A, single chain Fv: scFv, asparaginase B, tetra-cell adhesion molecule, B3(Fv)PE38, chloramphenicol acetyltransferase, neopullulanase, interleukin-1, interleukin-32, anti-B-lymphocyte antigen CD20, and anti-tumor necrosis factor alpha.

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