# US 2013/0280784 A1 · Escherichia coli expression system for producing mature human tyrosinase and a producing method thereof

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- Number: US 2013/0280784 A1
- Document ID: US20130280784A1
- Status: Published application
- Kind: A1
- Application number: 13/615,612
- Filed: 2012-09-14
- Published: 2013-10-24
- Earliest claimed: 2012-04-18
- Assignees: Providence University
- Inventors: Shann-Tzong JIANG; Gen-Hung CHEN
- Web page: https://patentgrep.com/patent/US20130280784A1

## CPC

- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/485: {Exopeptidases (3.4.11-3.4.19)}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12Y 114/18001: Tyrosinase (1.14.18.1)
- C12Y 304/11018: Methionyl aminopeptidase (3.4.11.18)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

An E. coli expression system for producing mature human tyrosinase is provided and includes an E. coli host, which has a trait for expressing endogenous methionyl aminopeptidase in cytoplasm, and an expression vector transformed into the E. coli host. The expression vector has a replication origin sequence of an E. coli, and the expression vector includes an inducible promoter and a DNA fragment of human tyrosinase having a sequence referenced as SEQ ID NO:3, wherein a 5′ end of the DNA fragment is constructed at a restriction enzyme NdeI recognition site located at the downstream of the inducible promoter.

## Claims

### Claim 1

An E. coli expression system for producing mature human tyrosinase, comprising:
an E. coli host having a trait for expressing endogenous methionyl aminopeptidase in cytoplasm; and
an expression vector transformed into the E. coli host, wherein the expression vector has a replication origin sequence of an E. coli, and the expression vector includes:
an inducible promoter; and
a DNA fragment of human tyrosinase having a sequence referenced as SEQ ID NO:3, wherein a 5′ end of the DNA fragment is constructed at a restriction enzyme NdeI recognition site located at the downstream of the inducible promoter.

### Claim 2 (depends on 1)

The expression system of claim 1, wherein the E. coli host is a BL21(DE3) strain, and the inducible promoter is a T7 promoter.

### Claim 3 (depends on 2)

The expression system of claim 2, wherein the expression vector originates from a pET23a(+) plasmid, and a 3′ end of the DNA fragment is constructed at a restriction enzyme XhoI recognition site located in the downstream of the inducible promoter.

### Claim 4 (depends on 1)

A method for producing mature human tyrosinase, comprising:
culturing an E. coli transformant in a liquid broth, wherein the E. coli transformant has an expression system of claim 1;
inducing the inducible promoter during a mid-log phase of the E. coli transformant at a temperature of not lower than 30° C. which the E. coli transformant can normally grow;
overexpressing recombinant proteins as inclusion bodies, wherein the recombinant proteins will then be hydrolyzed in cytoplasm of the E. coli transformant by an endogenous methionyl aminopeptidase to form a denatured mature human tyrosinase; and
refolding the recombinant proteins into a mature human tyrosinase in an active form.

### Claim 5 (depends on 4)

The method of claim 4, wherein the E. coli transformant is a BL21(DE3) strain, and the E. coli transformant comprises an expression vector having a T7 promoter.

### Claim 6 (depends on 5)

The method of claim 5, wherein the inducing step takes 3 to 12 hours.

### Claim 7 (depends on 5)

The method of claim 5, wherein the inducing step takes 9 hours.

### Claim 8 (depends on 5)

The method of claim 5, wherein the temperature of the inducing step is not lower than 37° C.

### Claim 9 (depends on 5)

The method of claim 5, wherein the refolding step comprises:
dissolving the inclusion bodies in a solubilization buffer containing 1% of sodium lauryl sulfate and 0.5% of 2-mercaptoethanol; and
refolding the inclusion bodies in a buffer containing copper ions.

### Claim 10 (depends on 9)

The method of claim 9, further comprising:
purifying the recombinant proteins by gel filtration chromatography after the refolding step of the recombinant proteins.