Protein or peptide printing method, protein array or peptide array, and functional protein or functional peptide identification method

Patent metadata

Field	Value
Publication number	US-9844764-B2
Application number	US-201313772835-A
Country	US
Kind code	B2
Filing date	Feb 21, 2013
Priority date	Aug 27, 2010
Publication date	Dec 19, 2017
Grant date	Dec 19, 2017

Abstract

The present invention relates to a protein or peptide printing method, comprising (a) a step for preparing nucleic acids and a cell-free protein synthesis system in an engraved plate composed of microscopic grooves having a specific opening shape, (b) a step for superimposing a substrate on the engraved plate so as to contact a protein or peptide to be synthesized in the microscopic grooves, and (c) a step for synthesizing the protein or peptide from the nucleic acids using the cell-free protein synthesis system in the microscopic grooves, and immobilizing the protein or peptide on the substrate along the specific opening shapes of the microscopic grooves.

First claim

The invention claimed is:

1. A protein or peptide printing method, comprising:
(a) preparing a mixture of a plurality of types of DNA, diluting the mixture to be dispensed so that there is 0 or 1 DNA molecule contained in each of microscopic grooves having a specific opening shape on an engraved plate, amplifying the DNA in each of the microscopic grooves, and adding a cell-free protein synthesis system, and a biotinylated complex of puromycin and nucleotide to each of the microscopic grooves,
(b) superimposing an avidin-labeled substrate on the engraved plate so as to contact a protein or peptide to be synthesized in the microscopic grooves, and
(c) synthesizing the protein or peptide from the nucleic acids using the cell-free protein synthesis system in the microscopic grooves, and immobilizing the protein or peptide on the substrate along the specific opening shapes of the microscopic grooves.

2. The protein or peptide printing method according to claim 1 , wherein the biotinylated puromycin derivative is a compound represented by the following general formula (1):

(wherein, Z represents a group represented by the following formula (2), (3) or (4):

(wherein, at least one of X 1 and X 2 represents a group represented by the following formula (5), and the other is a fluorescent group or hydrogen atom, and * represents a binding site:

(wherein, * represents a binding site))).

3. The protein or peptide printing method according to claim 2 , wherein Z is a group represented by the formula (2).

4. The protein or peptide printing method according to claim 1 , wherein the cell-free protein synthesis system consists of independently purified factors required for protein synthesis.

5. A protein or peptide printing method, comprising:
(a) preparing a mixture of: a plurality of types of DNA beads in each of which a single type DNA is immobilized on the beads through avidin-biotin or streptavidin-biotin binding, a cell-free protein synthesis system, a biotinylated complex of puromycin and nucleotide, in an engraved plate composed of microscopic grooves having a specific opening shape, a diameter of the microscopic grooves being 1 to 2 times a diameter of the beads, and a depth of the microscopic grooves being 1 to 2 times a diameter of the beads,
(b) superimposing an avidin-labeled substrate or a streptavidin-labeled substrate on the engraved plate so as to contact a protein or peptide to be synthesized in the microscopic grooves, and
(c) synthesizing the protein or peptide from the DNA using the cell-free protein synthesis system in the microscopic grooves, and immobilizing the protein or peptide on the avidin-labeled substrate or the streptavidin-labeled substrate along the specific opening shapes of the microscopic grooves.

6. The protein or peptide printing method according to claim 5 , wherein, in (a), the DNA is labeled with biotin, and are immobilized by magnetic beads that have been labeled with streptavidin.

7. A functional protein or functional peptide screening method, comprising:
(a) preparing a mixture of: a plurality of types of DNA beads in each of which a single type DNA is immobilized on the beads, and a cell-free protein synthesis system, in an engraved plate composed of microscopic grooves having a specific opening shape, a diameter of the microscopic grooves being 1 to 2 times a diameter of the beads, and a depth of the microscopic grooves being 1 to 2 times a diameter of the beads,
(b) superimposing a substrate on the engraved plate so as to contact a protein or peptide to be synthesized in the microscopic grooves,
(c) synthesizing the protein or peptide from the DNA using the cell-free protein synthesis system in the microscopic grooves, and immobilizing the protein or peptide on the substrate, thereby manufacturing a protein or peptide array, and
(d) carrying out functional screening using the protein or peptide array.

8. A method of manufacturing a set of protein array or a peptide array and an engraved plate, the method comprising:
providing the engraved plate having a plurality of microscopic grooves thereon and a substrate to be superimposed to the engraved plate; and
performing the protein or peptide printing method according to claim 1 .

9. The functional protein or functional peptide screening method according to claim 7 ,
wherein, the functional screening in (d) is measuring the activity of the protein or the peptide immobilized on the substrate.

10. The functional protein or functional peptide screening method according to claim 9 , further comprising
(e) performing a nucleotide sequence analysis on the DNA corresponding to the protein or peptide of which the activity is measured, thereby identifying the functional protein or the functional peptide.

Assignees

• Univ Tokyo
• Nippon Kogaku Kk
• Univ Tokyo

Inventors

• Ichiki Takanori
• Biyani Manish
• Shiono Hirofumi

Classifications

• B01J19/0046 Primary

  Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C07K17/14 Primary

  Peptides being immobilised on, or in, an inorganic carrier · CPC title

• C07K2319/21

  containing a His-tag · CPC title