Protein extraction method without lysing cells

Patent metadata

Field	Value
Publication number	US-11459571-B2
Application number	US-202117229546-A
Country	US
Kind code	B2
Filing date	Apr 13, 2021
Priority date	Oct 13, 2020
Publication date	Oct 4, 2022
Grant date	Oct 4, 2022

Abstract

A method for extracting protein without lysing cells includes: step 1: combining a penetrating peptide CPP gene having a sequence as set forth in SEQ ID NO: 1 and a bacterial lyase T4L gene having a sequence as set forth in SEQ ID NO: 2 through a flexible connecting peptide GGGGS gene having a sequence as set forth in SEQ ID NO: 3 to form a fusion enzyme CPP-T4L gene having a sequence as set forth in SEQ ID NO: 4; step 2: inserting the fusion enzyme CPP-T4L gene, and obtaining a recombinant host strain; step 3: cloning a target protein expression gene, and then constructing a recombinant expression strain; step 4: first inducing the expression of the target protein expression gene; starting the expression of the fusion enzyme CPP-T4L gene, and releasing a target protein; step 5: collecting cell lysis supernatant to recover the target protein.

First claim

What is claimed is:

1. A method for extracting a target recombinant protein from a recombinant host strain, comprising:
step 1: combining a penetrating peptide CPP gene (SEQ ID NO:1), and a bacterial lyase T4L gene (SEQ ID NO:2) by a flexible linker peptide gene (SEQ ID NO: 3) to form a fusion enzyme encoded by CPP-T4L gene (SEQ ID NO:4);
step 2: inserting the CPP-T4L gene into an expression vector, and then transferring the expression vector into an expression strain to obtain a recombinant host strain;
step 3: inserting a target protein expressing gene into a target protein expression vector, and then into said recombinant host strain;
step 4: in an expression system comprising said recombinant host strain,
first, inducing the target protein expressing gene and allowing for complete target gene expression;
second, adding an IPTG (isopropyl P-D-1-thiogalactopyranoside) solution as a lysis inducer, to the expression system to start releasing the expressed target protein in said recombinant host strain into solution; and
step 5: filtering the expression system solution and collecting the supernatant containing the recombinant target protein.

2. The method according to claim 1 , wherein in step 2, the vector comprising the CPP-T4L gene is Escherichia coli expression vector pGS21a, and the recombinant host strain is Escherichia coli BL21*(DE3).

3. The method according to claim 1 , wherein in step 2, the vector comprising the CPP-TL4 gene is transferred into the recombinant host strain by calcium chloride method.

4. The method according to claim 1 , wherein in step 3, the target protein expression vector is a pBAD/His vector.

5. The method according to claim 1 , wherein the target protein is lysostaphin, and arabinose is added in step 4 to induce the expression of the target protein.

6. The method according to claim 5 , wherein in step 4, arabinose is added at a final concentration of 0.1 mM to induce the expression of target protein.

7. The method according to claim 1 , wherein in step 4, the IPTG solution has a final concentration of 1 mM and is added to start the expression of the fusion enzyme.

Assignees

• Zha Jian
• Liu Zhiqiang
• Gong Guoli
• Sun Runcong
• Univ Shaanxi Science & Tech

Inventors

• Zha Jian
• Liu Zhiqiang
• Gong Guoli
• Sun Runcong

Classifications

• C12N15/62 Primary

  DNA sequences coding for fusion proteins · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C07K2319/10

  containing a tag for extracellular membrane crossing, e.g. TAT or VP22 · CPC title

• C12N9/50

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title