Method for quantification of amino acids using cell-free protein synthesis system

Patent metadata

Field	Value
Publication number	US-11519918-B2
Application number	US-201816633805-A
Country	US
Kind code	B2
Filing date	Jul 24, 2018
Priority date	Jul 24, 2017
Publication date	Dec 6, 2022
Grant date	Dec 6, 2022

Abstract

The present invention relates to a method for quantification of amino acids using a cell-free protein synthesis system. Specifically, the present invention relates to a method for quantification of amino acids, comprising: (a) preparing a reaction mixture for cell-free protein synthesis without target amino acids; (b) performing cell-free protein synthesis by mixing an assay sample containing target amino acids with the reaction mixture for cell-free protein synthesis; (c) measuring the signal intensity of the synthesized protein; and (d) calculating the concentration of the target amino acids by comparing the measured signal intensity with the standard concentration curve for each amino acid according to the protein signal produced using a standard sample, a method for providing information for diagnosing an amino acid metabolism-related disease using the same, a method for screening a material for prevention or treatment of an amino acid metabolism-related disease, and a method for screening a transaminase substrate. The method for quantification of amino acids according to the present invention can quantify amino acids in a short time at a low cost, and thus can be useful in various industries.

First claim

The invention claimed is:

1. A method for quantification of a target amino acid, comprising:
(a) preparing a reaction mixture for cell-free protein synthesis of a reporter protein comprising the target amino acid, wherein the reaction mixture comprises a mixture of amino acids for synthesizing the reporter protein which mixture does not include the target amino acid;
(b) performing cell-free protein synthesis by mixing an assay sample containing the target amino acid with the reaction mixture for cell-free protein synthesis;
(c) measuring the signal intensity of the synthesized reporter protein; and
(d) calculating the concentration of the target amino acid by comparing the measured signal intensity with a standard curve of the signal intensity of the reporter protein as a function of the concentration of the target amino acid in a standard sample.

2. The method of claim 1 , wherein the reaction mixture for the cell-free protein synthesis comprises a cell extract or a mixture of recombinant proteins.

3. The method of claim 2 , wherein the cell extract is an extract of E. coli , wheat germ, rabbit reticulocytes, yeast, Chinese Hamster Ovary cells, or HeLa cells.

4. The method of claim 2 , wherein the recombinant protein is at least one selected from the group consisting of an initiation factor, elongation factor, release factor, termination factor, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, RNA polymerase, and ribosome.

5. The method of claim 1 , wherein the reaction mixture for the cell-free protein synthesis comprises a polynucleotide encoding a reporter protein.

6. The method of claim 5 , wherein the reporter protein is a super-folder green fluorescent protein (sfGFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCherry fluorescent protein, lactamase, galactosidase, horseradish peroxidase (HRP), or glucose oxidase.

7. The method of claim 1 , wherein the assay sample is derived from at least one selected from the group consisting of a feed, food or chemical substance.

8. The method of claim 1 , wherein the assay sample is isolated from a living organism.

9. The method of claim 8 , wherein the assay sample is at least one selected from the group consisting of blood, plasma, serum, cancer tissue, and cancer cell.

10. The method of claim 8 , wherein the assay sample is heat treated and filtered.

11. The method of claim 8 , wherein the assay sample is isolated from a patient with an amino acid metabolism-related disease.

12. The method of claim 11 , wherein the amino acid metabolism-related disease is an amino acid metabolic disorder or cancer.

13. The method of claim 12 , wherein the amino acid metabolic disorder is at least one selected from the group consisting of arginase deficiency, cystinuria, maple syrup urine disease, hyperlysinemia, homocystinuria, hypermethioninemia, and phenylketonuria.

14. The method of claim 1 , wherein the method is used to quantify the target amino acid at a concentration of 10 nM to 150 uM.

15. A method for screening a substance for prevention or treatment of an amino acid metabolism-related disease, comprising:
(a) treating a candidate material for prevention or treatment of an amino acid metabolism-related disease with an assay sample isolated from a living organism;
(b) measuring the concentration of a target amino acid contained in the assay sample treated with the candidate material according to the method of claim 1 ; and
(c) comparing the amino acid concentration of step (b) with a normal control group.

16. The method of claim 15 , wherein the amino acid metabolism-related disease is an amino acid metabolic disorder or cancer.

17. The method of claim 16 , wherein the amino acid metabolic disorder is at least one selected from the group consisting of arginase deficiency, cystinuria, maple syrup urine disease, hyperlysinemia, homocystinuria, hypermethioninemia, and phenylketonuria.

18. A method for screening a transaminase substrate, comprising:
(a) reacting a transaminase with a mixture of a candidate amine donor substrate and amine acceptor substrate;
(b) measuring the concentration of a target amino acid contained in the reaction solution after completion of the reaction, wherein the measuring of the amino acid concentration is performed by the method of claim 1 ; and
(c) comparing the amino acid concentration of step (b) with a control group.

19. A method for screening a transaminase, comprising:
(a) reacting a candidate enzyme with a mixture of an amine donor substrate and amine acceptor substrate;
(b) measuring the concentration of a target amino acid contained in the reaction solution after completion of the reaction, wherein the measuring of the amino acid concentration is performed by the method of claim 1 ; and
(c) comparing the amino acid concentration of step (b) with a control group.

Assignees

• Iac In Nat Univ Chungnam

Inventors

• Kim Dong-Myung
• Yoo Tae Hyeon
• Lee Kyung-Ho
• Jang Yeon-Jae

Classifications

• G01N33/6893 Primary

  related to diseases not provided for elsewhere · CPC title

• G01N33/6812 Primary

  Assays for specific amino acids · CPC title

• G01N33/6806 Primary

  Determination of free amino acids · CPC title

• G01N2333/91188

  transferring nitrogenous groups (2.6) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title