CN104140960A - Application of cyclodextrin to reduce cytotoxicity in prokaryotic soluble expression system - Google Patents
Application of cyclodextrin to reduce cytotoxicity in prokaryotic soluble expression system Download PDFInfo
- Publication number
- CN104140960A CN104140960A CN201410363908.9A CN201410363908A CN104140960A CN 104140960 A CN104140960 A CN 104140960A CN 201410363908 A CN201410363908 A CN 201410363908A CN 104140960 A CN104140960 A CN 104140960A
- Authority
- CN
- China
- Prior art keywords
- cyclodextrin
- expression
- cytotoxicity
- protein
- expression system
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicinal Preparation (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the application of cyclodextrin to reduce cytotoxicity in a prokaryotic soluble expression system. According to the method, the cytotoxicity caused by soluble expression ribonuclease is avoided, the cytotoxicity and growth inhibition on Escherichia coli BL21-onconase from IPTG are reduced by addition of cyclodextrin, the influence on cell growth from soluble expression protein is also reduced, and expression of the soluble protein is added to lay a foundation for separating and purifying medical protein in the next step.
Description
technical field:
The present invention discloses a kind of cyclodextrin and in prokaryotic soluble expression system, reduces Cytotoxic purposes, the cytotoxicity that solution is produced due to solubility expression foreign protein (as frog's egg rnase), increase the output with biological activity protein, belong to biological medicine technology field.
Background technology
Procaryotic cell expression system, has that method is simple, product is convenient to purifying and the easy advantage such as evaluations, is the expression system of structure genetic engineering bacterium first-selection.In gene recombination technology, the exogenous gene expression product that the intestinal bacteria of take are host cell is often because the unfavorable cytotoxicity that produces of environment, suppress on the one hand the growth of cell, can make on the other hand foreign gene form in vivo the protein aggregate (inclusion body) of non-activity.Making the protein of non-activity become activated process is protein renaturation.In document, once reported multiple protein renaturation technology, but result not very good.
Method for solving cytotoxicity expression product, generally can adopt three kinds of methods, and the one, with eukaryotic expression system, but because complicacy and the fermentation time of eukaryotic expression system are relatively long, limit it and further apply; The 2nd, the expression method of employing gene fusion, soon the encoding gene of cytotoxicity product and other gene order link together and express, and then product is sheared, obtain having Cytotoxic product, but because purge process is loaded down with trivial details, cause its output not high; The 3rd, still utilize prokaryotic system, by inclusion body form, through renaturation, obtain recombinant protein, and renaturing inclusion bodies is the difficult problem in protein purification field always.
summary of the invention
The invention provides a kind of cyclodextrin and reduce Cytotoxic purposes in prokaryotic soluble expression system, using cyclodextrin as mini-chaperone, add in the genetic engineering bacterium BL21-Onconase substratum of solubility expression rnase (onconase), Cytotoxic impact while investigating it on e. coli bl21-onconase solubility expression Onconase, thus a kind of method that improves solubility expression foreign protein in protokaryon by reducing cytotoxicity is provided.
The present invention reduces Cytotoxic purposes by the fermenting experiment of e. coli bl21-onconase being shown to cyclodextrin in prokaryotic soluble expression system, and specific operation process is as follows:
1) picking mono-clonal BL21-Onconase is inoculated in 5ml LB substratum (100g/ml Ampicillin), and 37 ℃ of overnight incubation, by 1.2%(v/v) be inoculated in 100ml LB substratum (100 g/ml Ampicillin), 37 ℃ of cultivations, as the OD of bacterium liquid
600be 0.6 o'clock, add 0.1 mM IPTG and 1.0% beta-cyclodextrin, 30 ℃ of abduction delivering 4h;
2), after abduction delivering finishes, centrifugal (6000 rpm * 10 min), collects thalline;
3) thalline is in 20 mM, in pH 6.5 PBS, repetitive scrubbing is twice, centrifugal, be resuspended in isopyknic 20 mM, in pH 6.5 PBS, add PMSF(100 μ g/ml Virahol), adopt the broken thalline of sonioation method under multigelation cracking and ice bath, centrifugal (15000 rpm * 30 min), collect supernatant liquor, carry out 12% SDS-PAGE analysis, result Gel imaging and quantitative analysis software analysis, the growing state that represents cell with precipitation weight, the ratio that target protein expression amount accounts for full bacterium soluble proteins amount is index, compare analysis, the results are shown in Figure 1 and Fig. 2.
Fig. 1 result shows, is not adding cyclodextrin, and when IPTG concentration increases gradually from 0.1-1mM, the weight of thalline is reduced to 3.5 g/L from 4.5 g/L, illustrates that the solubility expression of IPTG induction produces cytotoxicity; Add after beta-cyclodextrin constant in IPTG concentration, as 0.1 mM, when the content of cyclodextrin is increased to 1.0% from 0.2%, the weight of thalline is increased to 7.2 g/L from 5.1 g/L, show to add cyclodextrin to increase the weight of thalline, reduce cytotoxicity and growth-inhibiting that IPTG causes, promoted Growth of Cells.
Fig. 2 result shows, at 1.0% beta-cyclodextrin, under 0.1mM IPTG, the onconase solubility expression amount in BL21-onconase cell is the highest, is 4.7 times of control group.Small molecules chemical chaperone not only can reduce cytotoxicity and promote expression of recombinant proteins, and the component of the onconase that can significantly improve expression in soluble proteins.In a word, cyclodextrin can reduce the cytotoxic effect that IPTG induction produces.
positively effect of the present invention is:by adding cyclodextrin, not only reduce IPTG to making cytotoxicity and the growth-inhibiting of e. coli bl21-onconase, and reduce the impact that causes cell growth due to solubility expression albumen, increase the expression of soluble proteins, for next step is laid a good foundation by separation and purification pharmaceutical protein.
accompanying drawing explanation:
The impact of Fig. 1 cyclodextrin on thalline weight;
The impact of Fig. 2 cyclodextrin on protein content.
Embodiment
embodiment 1
The source of frog rnase (onconase) has two kinds, and the one, from wood frog ovum, extract, but because source is limited, and in leaching process, the normal organic solvent that adopts can affect its biological activity, has limited its application; The 2nd, adopt gene engineering method to build engineering bacteria, in prokaryotic expression, conventionally adopt pFlag-CTS expression system, carrier has the OMPA signal peptide of methionine(Met) (M) guiding, at the N of original expression product end, it can guide expression product to arrive the pericentral siphon chamber of cell, and in cell by enzymic hydrolysis OMPA, produce and to have activated protein.Also be based on this reason, in cell, can produce cytotoxicity, affect Growth of Cells the further expression that affects foreign gene, rare to expression product under Routine Test Lab expression condition, Western blot detects and usually shows feminine gender.The present invention will provide a kind of cyclodextrin to reduce Cytotoxic purposes in prokaryotic soluble expression system, not only reduce cytotoxicity, and improve the expression of target protein.
The genetic engineering bacterium BL21-Onconase that expresses rnase is reached at 0.6 o'clock at 37 ℃ of OD600 that are cultured to bacterium liquid, add 0.1 mM IPTG and 1.0% beta-cyclodextrin, 30 ℃ of abduction delivering 4h; Collect thalline; Bacterial cell disruption, collects supernatant liquor, carries out 12% SDS-PAGE analysis, and result Gel imaging and quantitative analysis software analysis represents the growing state of cell to precipitate weight, and the ratio that target protein expression amount accounts for full bacterium soluble proteins amount is index.By Fig. 1 result, shown, do not adding cyclodextrin, when IPTG concentration increases gradually from 0.1-1mM, the weight of thalline is reduced to 3.5 g/L from 4.5 g/L, illustrates that the solubility expression of IPTG induction produces cytotoxicity; Add after beta-cyclodextrin constant in IPTG concentration, as 0.1mM, when the content of cyclodextrin is increased to 1.0% from 0.2%, the weight of thalline is increased to 7.2 g/L from 5.1 g/L, show to add cyclodextrin to increase the weight of thalline, reduce cytotoxicity and growth-inhibiting that IPTG causes, promoted Growth of Cells.
Fig. 2 result shows, at 1.0% beta-cyclodextrin, under 0.1mM IPTG, the onconase solubility expression amount in BL21-onconase cell is the highest, is 4.7 times of control group.Small molecules chemical chaperone not only can reduce cytotoxicity and promote expression of recombinant proteins, and the component of the onconase that can significantly improve expression in soluble proteins.
Conclusion: cyclodextrin can reduce the cytotoxic effect that IPTG induction produces.
embodiment 2
The single-chain antibody (ScFv) with activity of glutathione peroxidase of take is example, has built the prokaryotic soluble expression system that intestinal bacteria Rosetta contains recombinant plasmid pPelB-ScFv, cell concentration OD during expressing protein
600=0.6, inductor IPTG concentration 1.0 mM, induction time is 5 h, and the weight in wet base of result thalline in 1 L nutrient solution is 1.7 g, and in cellular lysate liquid supernatant, total protein concentration is 57 mg, and single-chain antibody shared ratio in lysate supernatant liquor is 10%; In the identical situation of above-mentioned condition, add 1.0% beta-cyclodextrin, result is that the weight in wet base of thalline in 1 L nutrient solution is 3.3 g, and in cellular lysate liquid supernatant, total protein concentration is 120 mg, and single-chain antibody shared ratio in lysate supernatant liquor is 22%.
Conclusion: by adding cyclodextrin, reduced the cytotoxicity that IPTG induction causes, maintained the production of cell, and promoted the expression of albumen.
embodiment 3
During ethanol dehydrogenase II (adhII) prokaryotic soluble expression, select pET-32a (+) expression vector, built recombinant plasmid pET-adhII, transform e. coli bl21 (DE3), obtain BL21-adhII genetic engineering bacterium, culturing bacterium is to cell concentration OD
600=0.6, inductor IPTG concentration 0.5 mM, induction time is 5 h, and the weight in wet base of result thalline in 1 L nutrient solution is 2.0 g, and in cellular lysate liquid supernatant, total protein concentration is 69 mg, and single-chain antibody shared ratio in lysate supernatant liquor is 12%; In the identical situation of above-mentioned condition, add 0.5% beta-cyclodextrin, result is that the weight in wet base of thalline in 1 L nutrient solution is 3.9g, and in cellular lysate liquid supernatant, total protein concentration is 150 mg, and single-chain antibody shared ratio in lysate supernatant liquor is 31%.
Conclusion: by adding cyclodextrin, reduced the cytotoxicity that IPTG induction causes, maintained the production of cell, and promoted the expression of albumen.
Claims (1)
1. cyclodextrin reduces Cytotoxic purposes in prokaryotic soluble expression system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410363908.9A CN104140960B (en) | 2014-07-29 | 2014-07-29 | Application of cyclodextrin to reduce cytotoxicity in prokaryotic soluble expression system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410363908.9A CN104140960B (en) | 2014-07-29 | 2014-07-29 | Application of cyclodextrin to reduce cytotoxicity in prokaryotic soluble expression system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104140960A true CN104140960A (en) | 2014-11-12 |
| CN104140960B CN104140960B (en) | 2017-05-03 |
Family
ID=51850301
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410363908.9A Expired - Fee Related CN104140960B (en) | 2014-07-29 | 2014-07-29 | Application of cyclodextrin to reduce cytotoxicity in prokaryotic soluble expression system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104140960B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1521183A (en) * | 2003-01-28 | 2004-08-18 | 大连百奥科技发展有限公司 | Method for high flux refolding renaturation of denaturation recombinant protein |
| CN101565448A (en) * | 2008-04-21 | 2009-10-28 | 张鹏 | Freeze-dried protein renaturation method |
-
2014
- 2014-07-29 CN CN201410363908.9A patent/CN104140960B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1521183A (en) * | 2003-01-28 | 2004-08-18 | 大连百奥科技发展有限公司 | Method for high flux refolding renaturation of denaturation recombinant protein |
| CN101565448A (en) * | 2008-04-21 | 2009-10-28 | 张鹏 | Freeze-dried protein renaturation method |
Non-Patent Citations (3)
| Title |
|---|
| LORETTA SHARMA1 等: "Influence of cyclodextrin ring substituents on folding-relatedaggregation of bovine carbonic anhydrase", 《FEBS》 * |
| 徐宏博等: "中国林蛙Onconase 样核糖核酸酶的基因克隆和表达", 《生物技术通报》 * |
| 范美音等: "中国林蛙细胞毒性核糖核酸酶的原核表达", 《生物技术》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104140960B (en) | 2017-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective | |
| Schlapschy et al. | Periplasmic chaperones used to enhance functional secretion of proteins in E. coli | |
| JP2016518833A5 (en) | ||
| WO2017106583A1 (en) | Cytoplasmic expression system | |
| Ukkonen et al. | Use of slow glucose feeding as supporting carbon source in lactose autoinduction medium improves the robustness of protein expression at different aeration conditions | |
| US10584347B2 (en) | Production of proteins in labyrinthulomycetes | |
| CN105238798A (en) | Expression gene, carrier, bacterial strain and preparation method of human methionine sulfoxide reductase A | |
| US20200270338A1 (en) | Expression constructs, host cells, and methods for producing insulin | |
| Mayer et al. | Lactose autoinduction with enzymatic glucose release: characterization of the cultivation system in bioreactor | |
| CN104140960A (en) | Application of cyclodextrin to reduce cytotoxicity in prokaryotic soluble expression system | |
| Lu et al. | Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations | |
| Samuelson et al. | Disulfide-bonded protein production in E. coli: Involvement of disulfide bond isomerase improves protein folding | |
| US9157085B2 (en) | Expression vector for expression of eukaryotic secretion leader sequence fused with a target polypeptide and methods of use thereof | |
| KR20160000979A (en) | Auto-Cell Concentration Recognition Auto-Inducible Expression System For Which Inducer Is Not Required and Use Thereof | |
| Naderi et al. | Crucial role of non-hydrophobic residues in H-region signal peptide on secretory production of l-asparaginase II in Escherichia coli | |
| CN111410693B (en) | CDK5 resistant nano antibody and application thereof | |
| ZHANG et al. | Advances in promoting soluble expression of recombinant protein in Escherichia coli | |
| Ha et al. | CO-EXPRESSION OF RECOMBINANT SINGLE CHAIN VARIABLE FRAGMENT RECOGNIZING BLOOD ANTIGEN FUSED WITH SUMO AND CHAPERONES IN Escherichia coli. | |
| Lai et al. | Identification of pyruvate carboxylase genes in Pseudomonas aeruginosa PAO1 and development of a P. aeruginosa-based overexpression system for α4-and α4β4-type pyruvate carboxylases | |
| US20060141571A1 (en) | Method for promoting cell growth and increasing the production of the expressed target gene products | |
| CN107058169B (en) | Application of sodium hydrosulfide in promoting expression of exogenous protein of escherichia coli | |
| CN105087725A (en) | Method for increasing expression level of soluble recombinant proteins in escherichia coli | |
| EP2128172B1 (en) | Preparation process of recombinant human p43 protein | |
| JP6909591B2 (en) | Production method of useful substances | |
| US11401311B2 (en) | Recombinant activin A precursor protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170503 Termination date: 20210729 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |