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Preparation of human basic fibroblast growth factor by utilizing Bacillus subtilis and endonuclease

A nucleic acid construct and intein technology, applied in the field of biology, can solve the problem that the factors of intein fragmentation are not very clear

Inactive Publication Date: 2020-06-05
DREAMTEC INTPROP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the factors that affect intein fragmentation are not well understood

Method used

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  • Preparation of human basic fibroblast growth factor by utilizing Bacillus subtilis and endonuclease
  • Preparation of human basic fibroblast growth factor by utilizing Bacillus subtilis and endonuclease
  • Preparation of human basic fibroblast growth factor by utilizing Bacillus subtilis and endonuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Expression vector construction and host cell transformation

[0093] Construction and Design of Escherichia coli / Bacillus subtilis Expression Shuttle Vector

[0094] pRB374 and pBR322 were used as the starting vector of E. coli / Bacillus subtilis expression shuttle vector [14]. Specifically, pECBS1 was constructed by the following modification steps: First, pRB374 (5.9 kb) was digested with EcoRI and PstI. The released fragment was replaced by the fragment obtained by digesting pBR322 (4.3 kb) with EcoRI and PstI, thereby forming the pECBSi vector. pECBSi was further digested with SalI and Dra, and the two sites were filled in with T4 DNA polymerase. Finally, these two sites were ligated together to form the pECBS1 shuttle vector.

[0095] Construction of bFGF expression vector

[0096] The construction method of the E. coli / Bacillus subtilis expression shuttle vector (pECBS1-H6-DnaE-bFGF) is as follows: a coding EcoRI-lac-operator (LacO)-ribosome bind...

Embodiment 2

[0099] Example 2: Expression of bFGF

[0100] shake flask culture

[0101] B. subtilis transformants were grown at 37°C (250 rpm) in 200 ml 2x LB medium supplemented with 25 μg / ml kanamycin [15]. When the A600 value reached 1.0, IPTG was added at a final concentration of 0.2 mM, and then 1 ml of culture samples were collected at 3-hour intervals for bFGF expression analysis. The cell pellet was resuspended in 200 μl of resuspension buffer (50 mM Tris-Cl, 200 mM EDTA, pH 8.0) and incubated on ice for 5 minutes. The mixture was then treated with 120 μl of lysozyme solution (10 mg / mL) for 20 minutes at 37°C. Then 80 μl of lysis buffer (10 mM EDTA, 10% Triton X-100 and 50 mM Tris-Cl, pH 8.0) was added. The tube containing the solution was gently inverted and then centrifuged at 14,800 rpm for 5 minutes. Cell lysate samples were analyzed by Western blot for bFGF protein expression.

[0102] In order to successfully express the soluble bFGF protein, the inventors also tested ...

Embodiment 3

[0111] Example 3: Purification and structural determination of bFGF protein

[0112] Cation exchange chromatography and heparin-agarose chromatography were used to purify bFGF. First, the protein concentration of the eluted fractions was measured using a Nanodrop Microvolume spectrophotometer. In addition, eluted fractions with significant readings (approximately 1 mg / ml) were pooled and dialyzed against 0.1x PB. Afterwards, the purified bFGF band was obtained by electrophoresis on a 10% SDS-PAGE gel stained with Coomassie brilliant blue R-250. The band containing bFGF protein in the SDS-PAGE gel was recovered for subsequent analysis by LC-MS.

[0113]The results of Western blot analysis showed that the soluble bFGF protein extracted from the lysate had the same molecular weight as the bFGF protein purchased from Thermofisher Scientific ( FIG. 4 ). Purified bFGF protein samples were subjected to N-terminal and C-terminal protein sequencing and MALDI-TOF mass determination b...

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Abstract

The invention relates to preparation of human basic fibroblast growth factor by utilizing Bacillus subtilis and endonuclease. Specifically, the invention provides a nucleic acid construct, which comprises an insert, the insert contains an affinity tag encoding a short peptide, a trans-splicing intein derived from Anabaena sp., and a polynucleotide sequence of an exogenous polypeptide, from the 5'end to the 3' end, wherein the short peptide affinity tag is used as an N-terminal extein of the trans-splicing intein, and the exogenous polypeptide is used as a C-terminal extein of the trans splicing intein. The invention also provides an expression vector and a host cell which contain the construct, and a method for generating and purifying exogenous proteins. According to the expression system and method of the present invention, the expression efficiency of exogenous proteins with biological activities can be significantly improved, the generation of inclusion bodies can be reduced, thepurification steps are simplified, the purification cost is greatly reduced, and the expression system and method is particularly suitable for large-scale culture.

Description

technical field [0001] The present invention relates to the field of biology, and generally to systems and methods for expressing exogenous polypeptides. Background technique [0002] Efficient and cost-effective expression of native forms of foreign polypeptides, especially cytokines, is increasingly important for the study of cell biology such as stem cells. Take human basic fibroblast growth factor (basic fibroblast growth factor, bFGF for short, also called FGF2) as an example. bFGF, a member of the fibroblast growth factor family, has diverse therapeutic uses in neurodegenerative diseases, heart disease, and difficult-to-heal wound lesions [1-3]. In addition, bFGF plays an important role in tissue development by inducing the proliferation of fibroblasts and stem cells [4-8], and it also plays an important role in the mass production of stem cells. However, the high production cost and low yield of bFGF protein currently hinder its commercial application in the pharmac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12P21/02C07K1/22C07K1/18C12R1/125
CPCC12N15/75C07K14/503C12N9/1252C12Y207/07007C12N15/52C12N15/70C07K14/50C12N15/62C12N15/63C07K1/22C07K2319/21C07K2319/92C12N2800/101
Inventor 钟树根邝纬阳
Owner DREAMTEC INTPROP LTD
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