# US 2021/0171925 A1 · Using Proteases to Control Restriction Enzyme Activity

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- Number: US 2021/0171925 A1
- Document ID: US20210171925A1
- Status: Published application
- Kind: A1
- Application number: 17/171,017
- Filed: 2021-02-09
- Published: 2021-06-10
- Earliest claimed: 2019-10-18
- Assignees: AbClonal Science, Inc.
- Applicants: ABclonal Science, Inc.
- Inventors: Zhenyu Zhu; Dapeng Sun; Aine Quimby; Michaela Shottes
- Web page: https://patentgrep.com/patent/US20210171925A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/37: involving peptidase or proteinase
- C12Y 304/21004: Trypsin (3.4.21.4)
- C12Y 304/2105: Lysyl endopeptidase (3.4.21.50)
- C12Y 304/21062: Subtilisin (3.4.21.62)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

Proteases are enzymes which hydrolyze protein enzymes, eliminating their activity. The present invention exploits the hydrolyzing activity of proteases including proteinase K, endoproteinase LysC and/or trypsin to control the activity of restriction enzymes and/or eliminate or reduce production of unwanted DNA or RNA fragments (known as star activity).

## Claims

### Claim 1

A process of inhibiting or preventing star activity by a restriction endonuclease comprising;
digesting an oligomer using a reaction mixture including the restriction endonuclease, buffer and oligomer;
including in the reaction mixture or adding proteinase K, endoproteinase LysC and/or trypsin in order to inhibit or prevent star activity by the restriction endonuclease; and
terminating the digestion by adding a stopping solution including a loading dye.

### Claim 2

(canceled)

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The process of claim 1 wherein the oligomer is lambda DNA.

### Claim 5 (depends on 1)

The process of claim 1 wherein the buffer is 20 mM Tris-HCl, pH 7.4, 40 mM KoAc, 10 mM MgCl2, 0.1 mM CaCl2; or 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mM MgCl2, 0.1 mM CaCl2.

### Claim 6 (depends on 1)

The process of claim 1 wherein the stopping solution includes 2.5% Ficoll®-400, 11 mM EDTA (pH 8.0), 3.3 mM Tris-HCl, 0.017% SDS, 0.015% bromophenol blue.

### Claim 7 (depends on 6)

The process of claim 6 wherein 6× the stopping solution is added.

### Claim 8

A process of determining the optimal relative concentrations to inhibit or prevent star activity by a restriction endonuclease, of the restriction endonuclease and a proteinase comprising:
(i) combining the restriction endonuclease, the proteinase, a buffer and an oligomer in order to achieve a starting concentration of each;
(ii) determining the star activity of the restriction endonuclease after a reaction time, and if there is any, diluting the reaction mixture and repeating the reaction for said reaction time;
(iii) repeating step (ii) until there is no star activity; and
(iv) determining the concentrations of restriction endonuclease and proteinase at which star activity ceases.

### Claim 9 (depends on 8)

The process of claim 8 wherein the proteinase are combined simultaneously with the restriction endonuclease.

### Claim 10 (depends on 8)

The process of claim 8 wherein the restriction endonuclease is EcoRI, NcoI or BamHI.

### Claim 11 (depends on 8)

The process of claim 8 wherein the proteinase is proteinase K, endoproteinase LysC or trypsin.

### Claim 12 (depends on 8)

The process of claim 8 wherein the oligomer is lambda DNA.

### Claim 13 (depends on 8)

The process of claim 8 wherein the buffer is: 20 mM Tris-HCl, pH 7.4, 40 mM KoAc, 10 mM MgCl2, 0.1 mM CaCl2; or 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mM MgCl2, 0.1 mM CaCl2; and, 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM CaCl2.

### Claim 14 (depends on 8)

The process of claim 8 wherein the restriction endonuclease activity is terminated by addition of STOP solution.

### Claim 15 (depends on 1)

The process of claim 1 wherein the buffer is 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM CaCl2.