Application of cellulase in soluable and secretion expression of recombinant protein in escherichia coli

A kind of Escherichia coli, recombinant protein technology, applied in the application field of cellulase protein

Active Publication Date: 2012-11-14
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Search results show that: its properties of soluble and secreted expression in Escherichia coli, and its application as a fusion

Method used

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  • Application of cellulase in soluable and secretion expression of recombinant protein in escherichia coli
  • Application of cellulase in soluable and secretion expression of recombinant protein in escherichia coli
  • Application of cellulase in soluable and secretion expression of recombinant protein in escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0059] Embodiment 1: Construction of high-efficiency expression vector of cellulase in Escherichia coli

[0060] 1. Strains and plasmids

[0061] Escherichia coli DH5α (E.coli DH5αLacZΔM15 hsdR recA)

[0062] Escherichia coli BL21(DE3) (E.coli BL21(DE3))

[0063] Escherichia coli BL21(DE3) / pLysS (E. coli BL21(DE3) / pLys)

[0064] Plasmid pET28a

[0065] The above strains and plasmids were purchased from Novagen.

[0066] 2. Enzymes and Reagents for Molecular Cloning

[0067] Restriction enzymes NcoI, BamHI; Pfu DNA polymerase; T4 ligase; protein molecular weight standards were purchased from MBI-Fermentas.

[0068] Agarose Gel DNA Purification Kit, DNA Fragment Purification Kit were purchased from OMEGA Ltd.

[0069] TIANprep Mini Plasmid Kit was purchased from TIANGEN.

[0070] Nucleic acid molecular weight standard 1Kb Marker was purchased from BioLab.

[0071] 3. Method

[0072] Using Bacillus sp.Z-16 genomic DNA as a template, use primers Cel-F / Cel-R to amplify t...

Embodiment 2

[0090] Example 2: Cellulase Cel is highly expressed and secreted in Escherichia coli

[0091] 1. SDS-PAGE reagent

[0092] Preparation of polyacrylamide gel:

[0093] Liquid A: acrylamide stock solution (30% w / v acrylamide, 0.8% w / v bisacrylamide), take 29.2g acrylamide, 0.8g

[0094] Bisacrylamide, ddH 2 O to 100mL, store at 4°C.

[0095] Solution B: Separating gel buffer, 1.5MTris-HCl (PH8.8) 0.4%SDS. Store at 4°C.

[0096] Solution C: stacking gel buffer, 1MTris-HCl (PH6.8) 0.4%SDS. Store at 4°C.

[0097] 10% ammonium persulfate: take 0.1g ammonium persulfate and add 1mL ddH 2 O, 4°C.

[0098] TEMED: purchased from Sigma, T8133

[0099] Mix the above solutions according to Table 2.1, prepare 12% separating gel and 5% stacking gel, pour into the gel plate, and use it for electrophoresis after polymerization.

[0100] Table 2.1 Polyacrylamide gel formula

[0101]

[0102] Electrophoresis buffer:

[0103] 10×Tris-glycine buffer: 144g glycine, 30g Tris, 10% SDS. ...

Embodiment 3

[0116] Example 3: Efficient and soluble expression of yeast sugar amidase (Png1p) in Escherichia coli by using the protein of the present invention

[0117] Preferably, the amino acid sequence of the catalytic domain of the cellulase is a fusion protein, and the yeast sugar amidase (Png1p) protein is taken as an example to verify the feasibility of the system of the present invention.

[0118] The N-terminal catalytic domain of Cellulase (abbreviated as Cel-CD) is the N-terminal sequence of Cellulase, as shown in SEQ ID NO: 2, consisting of 374 amino acids, and the theoretical length of the gene is 1125bp. Yeast glycoamidase (Png1p) is a protein with 363 amino acids, no signal peptide, and a molecular weight of 42.5kDa. The gene encoding Png1p is located on the left arm of yeast XVI chromosome and consists of 1092 nucleotide sequences. Png1p can be highly expressed in Escherichia coli, but they all exist in the form of inclusion bodies.

[0119] 1. Reagents:

[0120] Buffer ...

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Abstract

The invention discloses an application of cellulase in soluable and secretion expression of recombinant protein in escherichia coli. The protein sequence is an amino acid sequence represented by SEQ ID NO: 1-7, or a polypeptide sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 1-7, and the recombinant protein is antibacterial peptides or enzymes. The application of cellulase in soluable and secretion expression of recombinant protein in escherichia coli provides a foundation for preparing protein/ polypeptide drugs, various enzymes, diagnostic antigens, antibodies, vaccines and the like by using escherichia coli, and has important theoretical meanings and application values in protein expression study, enzymic preparations, protein drug production and the like.

Description

technical field [0001] The invention relates to the application of a cellulase protein, in particular to the application of a cellulase protein in soluble expression and secreted expression of recombinant protein in Escherichia coli. Background technique [0002] Every cell and every major part of the body has proteins involved. Therefore, protein molecules are the cornerstone of the development of modern biotechnology and biopharmaceutical industry. Protein has always been an important aspect of biological research. Obtaining a large number of purified protein samples is the basis for the study of protein physicochemical properties and functions, and the development of biopharmaceutical and enzyme preparation industries. [0003] In order to obtain a large number of recombinant proteins, people have developed a variety of protein expression systems, and the Escherichia coli expression system is widely used because of its simple operation, clear genetic background, mature t...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C12N9/78C07K14/435C07K14/605
Inventor 祁庆生王圣钧高冬芳
Owner SHANDONG UNIV
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