WO2015176641A1

WO2015176641A1 - Method for promoting soluble expression of recombinant extremely thermostable α-amylase

Info

Publication number: WO2015176641A1
Application number: PCT/CN2015/079267
Authority: WO
Inventors: 张毅; 彭帅英; 杨胜利
Original assignee: 中国科学院上海生命科学研究院
Priority date: 2014-05-21
Filing date: 2015-05-19
Publication date: 2015-11-26
Also published as: CN105087520B; CN105087520A

Abstract

Provided is a method for promoting soluble expression of recombinant extremely thermostable α-amylase, containing a co-expression of a gene encoding extremely thermostable α-amylase and a gene encoding the molecular chaperone protein Prefoldin; thus the soluble expression and enzyme activity of extremely thermostable α-amylase are improved.

Description

Method for promoting recombinant soluble heat-resistant α-amylase soluble expression

Technical field

The present invention is in the field of biotechnology; more specifically, the present invention relates to a method for promoting the soluble expression of recombinant heat-resistant alpha-amylase.

Background technique

Alpha-amylase, also known as starch 1,4-dextrinase, is an endonuclease that randomly cleaves α-1,4-glycosidic linkages in starch, glycogen, oligomeric or polysaccharide molecules. It produces maltose, oligosaccharides and glucose, and is one of the most widely used enzyme preparations in industrial production. At present, α-amylase has been widely used in many industries and pharmaceutical industries such as modified starch and starch sugar, baking industry, beer brewing, alcohol industry, fermentation and textile, and is an important industrial enzyme.

Thermostable alpha-amylase is one of the most commonly used enzyme preparations in the industry. The heat-resistant α-amylase commonly used in the industry today is derived from Bacillus licheniformis (BLA), and its optimum temperature is 90 ° C and needs to interact with Ca ²⁺ to maintain its thermal stability. However, the enzyme still has shortcomings: First, the thermal stability of BLA is still not high enough; secondly, the optimum pH of BLA is 7.0-8.0; again, BLA requires calcium ions to maintain its thermal stability.

Recombinant extreme heat-resistant α-amylase PFA is derived from the extremely thermophilic archaea P. furiosus, which has excellent enzymatic properties and enzymatic characteristics, and is far superior to the traditional heat-resistant α on the market in many key indicators. - Amylase. For example, its optimum temperature is close to 100 ° C, and it has extremely excellent heat resistance, and does not lose activity at 100 ° C for several hours (generally heat-resistant amylase such as BLA has a half-life of only a few minutes at nearly 100 ° C). At the same time, the activity and heat resistance of the extremely heat-resistant α-amylase PFA do not require additional calcium ions, and the optimum pH is acidic, which can greatly facilitate the subsequent steps in the industrial application process. In addition, the main component of the starch hydrolysate of the enzyme is malto-oligosaccharide, which is mainly G2-G7, and the monosaccharide (glucose) content is less than 2%. These advantages and characteristics suggest that recombinant PFA is expected to play an outstanding role in a variety of industrial production processes including high temperature liquefaction of starch, production of ultra high purity high maltose syrup, and textile printing and dyeing, and has extremely high potential industrial application value.

P.furiosus is an absolute anaerobic bacterium isolated from submarine volcanic hot springs. The optimum growth temperature is 100 °C [Haruyuki Atomi et al., Current Opinion in Biotechnology, 2011, 5(22): 618-626]. The culture is relatively complicated. It is difficult to achieve large-scale industrial cultivation, and a large amount of PFA preparation It can only be achieved by exogenous recombinant expression. So far, many domestic and foreign laboratories have studied the protein structure, enzymatic properties and recombinant expression of PFA. So far, there is no report suitable for the large-scale preparation of the enzyme. The results of the foreign research and the inventors' research on PFA found that when recombinant PFA is expressed in a large amount in E. coli, the expressed recombinant PFA is mainly an insoluble inclusion body, which is a downstream engineering of large-scale purification and preparation of the enzyme. Brought great inconvenience.

The present inventors conducted a relatively in-depth study on the recombinant extreme heat-resistant alpha-amylase PFA. First, a large amount of recombinant PFA was expressed in E. coli and it was found that recombinant PFA was mainly expressed in the form of insoluble inclusion bodies. Then, the method of purification and renaturation of PFA inclusion bodies was studied, and the purification method of alkali denaturation-heat-dilution renaturation was established, and recombinant PFA with high biological activity was obtained [Lisa Wang et al., J Ind Microbiol Biotechnol. 2007. (34): 187-192; Zhang Yi et al., Chinese invention patent: a preparation and purification method of thermophilic α-amylase, patent number: ZL2006 1 0025974.0]. In the further study on the stability of purified enzymes, it was found that a certain concentration of detergent had no effect on the enzymatic activity of recombinant PFA, and thus a method for the renaturation and purification of recombinant PFA inclusion bodies by detergent was established. Zhang Yi et al., Chinese invention patent: Refolding and purification method of recombinant extreme heat-resistant α-amylase, patent number: ZL200810039429.6].

Nevertheless, for recombinant enzymes, soluble expression is more applicable to the convenience of production and production and controllability of production costs. Due to the nature of the recombinant extreme heat-resistant alpha-amylase PFA, it is difficult to achieve soluble recombinant high expression of the enzyme in the prior art.

Therefore, there is a need in the art to further investigate expression systems and expression methods suitable for recombinant expression of the extreme heat-resistant alpha-amylase PFA.

Summary of the invention

It is an object of the present invention to provide a method for promoting the soluble expression of recombinant heat-resistant alpha-amylase.

In a first aspect of the invention, a method for recombinantly expressing an extreme thermostable alpha-amylase, the method comprising: co-expressing a gene encoding an extreme thermostable alpha-amylase with a gene encoding a chaperone protein Prefoldin, A soluble extreme heat-resistant alpha-amylase is obtained.

In a preferred embodiment, the co-expressed expression host comprises: a prokaryotic cell, a eukaryotic cell. Preferably, said prokaryotic cells include, but are not limited to, E. coli cells; said eukaryotic cells include but Not limited to yeast cells. Preferably, the expression host is an E. coli cell.

In another preferred embodiment, the molecular chaperone protein Prefoldin is derived from the extremely thermophilic archaea P. furiosus.

In another preferred embodiment, the extreme thermostable alpha-amylase is derived from the extremely thermophilic archaea P. furiosus.

In another preferred embodiment, the gene encoding the extreme thermostable alpha-amylase and the gene encoding the chaperone protein Prefoldin are co-expressed in E. coli cells.

In another preferred embodiment, the method of co-expression includes:

(1) Providing an expression vector comprising an expression cassette of an extreme heat-resistant α-amylase and an expression cassette of a molecular chaperone protein Prefoldin;

(2) The expression vector of (1) is transformed into Escherichia coli cells, and recombinant E. coli cells transformed with the expression vector are cultured to obtain a soluble extreme heat-resistant α-amylase.

In another preferred embodiment, the expression cassette containing the extreme thermostable alpha-amylase and the expression cassette of the chaperone protein Prefoldin are located in different expression vectors, respectively, or in the same expression vector.

In another preferred embodiment, the expression vector includes, but is not limited to, pT7473, pACYC or pCDF.

In another preferred embodiment, the extreme thermostable alpha-amylase is an extreme thermostable alpha-amylase PFA.

In another aspect of the invention, there is provided the use of the molecular chaperone protein Prefoldin for promoting the expression of recombinant extreme heat-resistant alpha-amylase.

In a preferred embodiment, the chaperone protein Prefoldin promotes soluble expression of an extremely thermostable alpha-amylase by co-expression with an extremely thermostable alpha-amylase.

In another aspect of the invention, a recombinant host cell comprising an expression cassette for an extreme thermostable alpha-amylase and an expression cassette for the chaperone protein Prefoldin is provided.

In a preferred embodiment, the host cell is an E. coli cell.

In another aspect of the invention, a kit for recombinant expression of an extreme thermostable alpha-amylase is provided, the kit comprising:

An expression vector containing an expression cassette and molecular chaperone egg of an extremely thermostable alpha-amylase White Prefoldin expression cassette.

In a preferred embodiment, the expression cassette containing the extreme thermostable alpha-amylase and the expression cassette of the chaperone protein Prefoldin are located in different expression vectors, respectively, or in the same expression vector.

In another preferred embodiment, the kit further comprises: E. coli cells.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

DRAWINGS

Figure 1. Effect of the molecular chaperone proteins Prefoldin and Hsp60 derived from P. furiosus on the soluble expression of recombinant heat-resistant amylase PFA.

A is the specific activity of PFA in the supernatant after crushing each strain, BL21(DE3) is the empty host strain, PFA is the recombinant PFA expressing strain, and PFA+pACYC is the strain which simultaneously transforms pACYC plasmid and PFA expression plasmid, PFA+pACYC - Prefoldin is a strain which simultaneously transforms the pACYC-Prefoldin plasmid and the PFA expression plasmid, and PFA+pACYC-Hsp60 is a strain which simultaneously transforms the pACYC-Hsp60 plasmid and the PFA expression plasmid.

B is the SDS-PAGE electrophoresis pattern of the centrifuged supernatant and precipitated samples after crushing each strain. The samples in each lane are as follows: M is the marker; 1-2 is the supernatant and precipitate after the breakage of the BL21 (DE3) cells, 3-4 For the recombinant PFA expression, the supernatant was disrupted and precipitated, and 5-6 was the same as the PACYC plasmid and the PFA expression plasmid, and the supernatant was disrupted and precipitated (PFA+pACYC), and 7-8 was used to transform pACYC-Prefoldin plasmid and PFA simultaneously. The plasmid-expressing cells were disrupted by supernatant and pellet (PFA+pACYC-Prefoldin), and 9-10 was a bacterial cell disrupted supernatant and pellet (PFA+pACYC-Hsp60) which simultaneously transformed the pACYC-Hsp60 plasmid and the PFA expression plasmid.

Figure 2. Effect of increased Prefoldin expression on the soluble expression of PFA.

A is the specific activity of PFA in the centrifuged supernatant of each strain. BL21(DE3) is an empty host strain, PFA is a recombinant PFA expressing strain, PFA+pACYC is a strain that simultaneously transforms pACYC plasmid and PFA expression plasmid, and PFA+pACYC-Prefoldin is a simultaneous transformation of pACYC-Prefoldin plasmid and PFA expression. The granulocyte strain, PFA+pCDF, is a strain which simultaneously transforms the pCDF plasmid and the PFA expression plasmid, and PFA+pCDF-Prefoldin is a strain which simultaneously transforms the pCDF-Prefoldin plasmid and the PFA expression plasmid.

B is the SDS-PAGE electrophoresis pattern of the supernatant and precipitate after crushing each strain. The samples in each lane are as follows: M is the marker; 1-3 is the PFA cell, the supernatant and the precipitated sample, and 4-6 is the PFA+pACYC- Prefoldin cells, supernatant and precipitated samples, 7-9 were PFA+pCDF-Prefoldin cells, supernatant and precipitated samples.

Figure 3. The recombinant PFA and the chaperone Prefoldin further enhance the soluble expression level of PFA when co-expressed in a plasmid. a. The specific activity of the recombinant PFA in the supernatant after the disruption of each recombinant bacterial cell. b. Protein SDS-polyacrylamide gel electrophoresis analysis results. M: protein molecular weight standard; 1-3: BL21 (DE3) whole bacteria, supernatant after crushing, precipitation of samples; 4-6: pT7473-PFA strain whole bacteria, crushed supernatant, precipitated samples; 7-9: pACYC- Whole strain of PFA strain, supernatant after crushing, precipitated sample; 10-12: whole strain of pT7473-PFA+pCDF-prefoldin strain, supernatant after crushing, precipitated sample; 13-15: whole strain of pT7473-PFA+pACYC-prefoldin strain , after the crushed supernatant, precipitated samples; 16-18pCDF-PFA strain whole bacteria, crushed supernatant, precipitated samples; 19-21: pACYC-prefoldin-PFA strain whole bacteria, crushed supernatant, precipitated samples; 22-24 : pCDF-prefoldin-PFA strain whole bacteria, the supernatant was crushed, and the sample was precipitated.

detailed description

The inventors have intensively studied and developed a method for promoting the soluble expression of recombinant heat-resistant α-amylase by repeated attempts of various expression systems and a large number of expression methods. The method is effective to increase the amount of soluble heat-expressing extreme heat-resistant alpha-amylase and to retain the biological activity of the enzyme well.

The present inventors have long been working on recombinant expression studies of extreme heat-resistant α-amylases, and tried various expression systems and various methods for promoting their soluble expression. For prokaryotic expression systems, the inventors investigated and explored whether co-expression of different chaperone proteins in bacteria would help to increase the soluble expression of recombinant PFA. The results showed that small chaotic protein (sHSP), HSP60 and other chaperone proteins could not effectively promote the soluble expression of extreme heat-resistant α-amylase. The inventors unexpectedly found in the experiment that when Prefoldin was co-expressed with PFA, the soluble expression of recombinant PFA was significantly improved.

Furthermore, the inventors examined the effect of the expression level of recombinant Prefoldin on the soluble expression of PFA. A method for significantly increasing the expression of Prefoldin was found, and a further increase in the expression level significantly promoted the soluble expression of recombinant PFA. This experimental result is of great significance for the future industrial production and application of extreme heat-resistant alpha-amylase (PFA).

Recombinant extreme heat-resistant alpha-amylase PFA

The amino acid sequences of the recombinant extreme thermostable alpha-amylase PFAs of the invention are known in the art.

As a preferred mode of the present invention, the recombinant extreme heat-resistant α-amylase PFA is: (a) a protein of the amino acid sequence of SEQ ID NO: 3; or (b) a sequence of the amino acid of SEQ ID NO: 3 Or a plurality of (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acid residue substitutions, deletions or additions, and having a recombinant heat-promoting alpha-amylase A biologically active fragment of a protein derived from (a) that is soluble in function; or (c) a protein of the amino acid sequence set forth in SEQ ID NO: 3.

The gene encoding the PFA protein is readily available, and in the case where the protein is known, those skilled in the art know how to obtain the gene encoding the PFA, for example, by PCR amplification from the genome, or by chemical synthesis.

Prefoldin

Prefoldin (PFD), also known as the GimC/Gim protein, is a hexamer chaperone protein complex composed of two subunits found in all eukaryotes and archaea. Prefoldin derived from an extremely thermophilic archaea consists of two alpha subunits and four beta subunits forming a protein matrix similar to the "jellyfish" shape, the structure of which consists of two beta-folded barrels, six long curly The tentacle stretches out and the top of the tentacle exposes the hydrophobic area.

The α subunit of the Prefoldin protein is: (a) a protein of the amino acid sequence of SEQ ID NO: 1; or (b) one or more (preferably 1-20) of the amino acid sequence of SEQ ID NO: 1. More preferably from 1 to 10, more preferably from 1 to 5) substitutions, deletions or additions of amino acid residues, and having (a) derived from (a) which promotes the soluble expression of recombinant heat-resistant α-amylase a biologically active fragment of a protein; or (c) a protein of the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, the Prefoldin protein beta subunit is: (a) a protein of the amino acid sequence of SEQ ID NO: 2; or (b) one or more (preferably 1-20) of the amino acid sequence of SEQ ID NO: 2. , preferably 1-10, more preferably 1-5) formed by substitution, deletion or addition of an amino acid residue, and having (a) derived from (a) promoting the soluble expression function of recombinant heat-resistant α-amylase Biological activity of the protein; or (c) the amino acid sequence of SEQ ID NO: 2 Sexual fragment.

The amino acid sequence of the alpha subunit of the Prefoldin protein can also be found in Genbank accession number: NP_578104. The amino acid sequence of the beta subunit of the Prefoldin protein can also be found in Genbank accession number: NP_578111.1.

The gene encoding the Prefoldin protein is readily available. In the case where the protein is known, one skilled in the art knows how to obtain the gene encoding Prefoldin, for example, by PCR amplification from the genome, or by chemical synthesis. For example, the gene encoding the subunit of Prefoldin protein alpha can be found in Genbank accession number: PF0375. The gene encoding the β subunit of the Prefoldin protein can be found in Genbank accession number: PF0382. Furthermore, sequences degenerate from the gene sequences obtained by the above amplification are also available.

Prefoldin is derived from the extremely thermophilic archaea P. furiosus, and it is uncertain in the art whether it contributes to the recombinant expression of proteins in prokaryotic expression systems.

Recombinant expression

The present invention also relates to an expression vector comprising the gene encoding the gene encoding the extreme thermostable alpha-amylase PFA and/or Prefoldin, and a host cell produced by genetic engineering transformation using the vector of the present invention.

The expression vector of the present invention is an expression vector suitable for expression by a host cell (expression host), which contains an expression cassette of the extreme heat-resistant α-amylase PFA and/or Prefoldin. As used herein, "expression cassette" refers to a gene expression system comprising all of the necessary elements required to express a polypeptide of interest (PFA and Prefoldin in the present invention), typically comprising the following elements: a promoter, a polypeptide encoding Gene sequences, terminators; in addition, optionally include signal peptide coding sequences, etc.; these elements are operably linked.

As a preferred mode of the invention, the expression vector includes, but is not limited to, pT7473, pACYC or pCDF. More preferably, the pCDF expression vector is used to express Prefoldin, which promotes an increase in the soluble extreme heat-resistant alpha-amylase PFA.

The expression vector is transformed into an expression host, and a recombinant expression host transformed with the expression vector is cultured to express a soluble extreme heat-resistant α-amylase PFA. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.

The host cell may be a prokaryotic cell, a eukaryotic cell or the like. a specific of the prokaryotic cell An example is E. coli.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

Materials and Methods

Strain and vector

Construction of pT7473-PFA expression vector: See Lisa Wang et al, J Ind Microbiol Biotechnol. 2007. (34): 187-192 for details.

Construction of pACYC-Prefoldin vector and pCDF-Prefoldin vector: extraction of pPFD plasmid (for the construction of pPFD plasmid, see Hui Chen et al., Biotechnol Lett. 2010. (32): 429-434), and two restriction endonucleases with NcoI and SalI. The gene expression fragment encoding Prefoldin was obtained by double digestion, and purified by agarose gel electrophoresis for purification. pACYCDuet-1 (Novagen) and pCDFDuet-1 plasmid (Novagen) were digested with NcoI and SalI, respectively, and recovered by agarose gel electrophoresis. The double-digested Prefoldin coding gene was mixed with the double-encapsulated pACYCDuet-1 and pCDFDuet-1 plasmids, and T4 DNA ligase was added thereto, and reacted at 16 ° C for 16 hours to obtain pACYC-Prefoldin vector and pCDF-Prefoldin vector, respectively.

Construction of pACYC-PFA: PFA with PshBI/XhoI enzyme at both ends was cloned into the corresponding site of pACYC plasmid.

Construction of pCDF-PFA: PFA with PshBI/XhoI enzyme at both ends was cloned into the corresponding site of the pCDF plasmid.

Construction of pACYC-Prefoldin-PFA: Pre-purification of pACYC-PFA was carried out, and Prefoldin with NcoI/SalI enzyme at both ends was cloned into the corresponding site of the plasmid to obtain pACYC-Prefoldin-PFA.

Construction of pCDF-Prefoldin-PFA: Pre-purification of pCDF-PFA was performed, and Prefoldin with NcoI/SalI enzyme at both ends was cloned into the corresponding site of the plasmid to obtain pCDF-Prefoldin-PFA.

Construction of pACYC-Hsp60 vector: The pET21-PfCPN plasmid containing the HSP60-encoding gene was extracted (for the construction of pET21-PfCPN plasmid, see Hua-you Chen et al., Journal of Basic Microbiology 2007. (47): 132-137), using Nde I and Xhol. I double digestion, HSP60 encoding gene DNA fragment Agarose gel electrophoresis was recovered and purified for use. The pACYCDuet-1 plasmid was digested with Nde I and Xhol I and ligated with the recovered HSP60-encoding gene DNA fragment to obtain a pACYC-Hsp60 vector.

The above expression vectors were transformed into Ecoli BL21 (DE3) strains alone or in different combinations to obtain the following recombinant strains, respectively:

An Ecoli BL21 (DE3) expression strain carrying the pT7473-PFA expression vector;

Ecoli BL21 (DE3) expression strain carrying the pACYC-Prefoldin vector;

Ecoli BL21 (DE3) expression strain carrying the pCDF-Prefoldin vector;

An Ecoli BL21 (DE3) expression strain carrying both pT7473-PFA and pACYC-Prefoldin vectors;

Ecoli BL21 (DE3) expression strain carrying both pT7473-PFA and pCDF-Prefoldin vectors.

An Ecoli BL21 (DE3) expression strain carrying both pT7473-PFA and pACYC-HSP60 vectors.

Ecoli BL21 (DE3) expression strain carrying the pACYC-Prefoldin-PFA vector.

Ecoli BL21 (DE3) expression strain carrying the pCDF-Prefoldin-PFA vector.

Ecoli BL21 (DE3) expression strain carrying pCDF-PFA.

Ecoli BL21 (DE3) expression strain carrying pACYC-PFA.

Main reagents and solutions

LB medium: 10 g NaCl, 5 g yeast powder, 10 g peptone dissolved in 1000 ml deionized water;

Ultrasonic disruption buffer: Tris.HCl (pH 8.0) 50 mM, EDTA 10 mM, NaCl 100 mM;

DNS reagent: 1g 3,5-dinitrosalicylic acid is dissolved in 20ml 2mol / L NaOH, add 30g potassium sodium tartrate, diluted to 100ml;

Copper-tartrate-carbonate (CTC): Slowly add 20% (w/v) sodium carbonate solution to the copper sulfate-tartaric acid solution and slowly stir until the final concentration of copper sulfate (pentahydrate) is 0.1% ( w/v), the final concentration of sodium potassium tartrate is 0.2% (w / v), the final concentration of sodium carbonate is 10% (w / v);

10% SDS: 10 g of SDS is dissolved in 100 ml of deionized water to prepare a 10% stock solution, which is diluted 10 times when used;

0.8N NaOH;

Folin-phenol reagent: purchased from the factory, the concentration is 2N;

0.15% (w/v) sodium deoxycholate (DOC);

72% (w/v) sodium trichloride (TCA);

Reagent A: Mix CTC, NaOH, SDS with water in equal amounts (by volume);

Reagent B: One volume of 2N Folin-phenol reagent was mixed with 5 volumes of deionized water.

Recombination of recombinant cells

The glycerol-preserving strain was inoculated first at -80 ° C in 3 ml of LB medium which was sterile but contained corresponding resistance, and cultured at 37 ° C, 250 rpm overnight. On the next day, the overnight cultured inoculum was inoculated with 1% of the inoculum in 200 ml of sterile LB containing the corresponding resistance for 2 h. After 2 h, IPTG was added to a final concentration of 0.1 mM for 4 h. After 4 hours, it was centrifuged at 5000 rpm for 30 min, the supernatant was discarded, and the cells were collected and the wet weight of the cells was measured.

BL21(DE3), BL21(DE3) containing pT7473-PFA recombinant plasmid, BL21(DE3) containing pACYC and pT7473-PFA recombinant plasmid, BL21 containing pACYC-Prefoldin and pT7473-PFA recombinant plasmid (DE3) were collected in the same manner. ), BL21 (DE3) containing pCDF and pT7473-PFA recombinant plasmid, and BL21 (DE3) strain containing pCDF-Prefoldin and pT7473-PFA recombinant plasmid.

Cell disruption

The cells were suspended with 0.1 g/ml of the viable cell suspension according to the weight of the wet cells, sonicated for 5 min, at intervals of 5 min, and then crushed for 5 min. All operations were carried out on an ice bath. The crushing operations of all samples were consistent. After crushing, centrifugation was carried out at 10,000 rpm for 30 min, and the supernatant and the precipitate were collected, and the precipitate was resuspended in the same volume of the disrupted solution and shaken well. The same volume (15 ul) of supernatant and precipitated SDS-PAGE protein electrophoresis were taken. Amylase activity was measured at 100 ° C after another sample was moderately diluted. The remaining samples were stored in a 4 ° C refrigerator for subsequent experiments.

Method for measuring high temperature amylase activity (DNS method)

The obtained supernatant was quantitatively diluted, and the supernatant activity was measured.

The quantitatively diluted enzyme solution was added to a 50 mmol/L sodium acetate solution containing 1% (w/v) starch to a final volume of 500 ul, reacted at 100 ° C for 15 min, and quickly placed in an ice water bath to terminate the reaction. Add 500ul of DNS reagent, boil in boiling water for 5 minutes, quickly put it into ice water to cool, add 5ml of water, Shake well and measure the absorbance at 546 nm using an ultraviolet-visible spectrophotometer. The reaction of glucose with DNS reagent was used as a standard curve. One enzyme activity unit is defined as the amount of enzyme that degrades starch to form 1 umol of reducing sugar in 1 minute.

Method for determining protein content (Folin-phenol method)

The obtained supernatant was quantitatively diluted, and the supernatant protein concentration was measured.

Add 0.1 ml of 0.15% (w/v) sodium dehydrocholate to 1 ml of sample, leave it at room temperature for 10 min, add 0.1 ml of 72% (w/v) TCA solution, mix well, centrifuge at 3000 rpm for 15 min, discard the supernatant. . 1 ml of deionized water was added to the precipitate, 1 ml of Reagent A was added, mixed well and left at room temperature for 10 min. 0.5 ml of reagent B was added and immediately mixed uniformly. After 30 minutes, the absorbance at 750 nm was measured by a spectrophotometer.

Example 1. Effect of Prefoldin and HSP60 on Soluble Expression of Extremely Thermostable Amylase PFA

The present inventors examined molecular chaperone proteins of various origins and examined whether they contribute to the soluble expression of recombinant PFA when they are co-expressed with the extreme thermostable amylase PFA. After screening, the scope of the study was finally determined: two molecular chaperone proteins, Prefoldin and Hsp60, derived from the extremely thermophilic archaea P. furiosus.

The coding sequences of Prefoldin and Hsp60 were cloned into the pACYC plasmid, respectively, and labeled as pACYC-Prefoldin and pACYC-Hsp60 plasmids, respectively. BL21 (DE3) host strain, BL21 (DE3) containing pT7473-PFA recombinant plasmid, and BL21 (DE3) strain containing pACYC and pT7473-PFA plasmids were used as controls. The above strain was taken out from a glycerol tube frozen at -80 ° C, inoculated into 3 ml of LB medium having corresponding resistance, and cultured overnight at 37 ° C on a shaker at 250 rpm. The overnight culture medium was transferred to 200 ml of LB medium containing the corresponding resistance at 37 ° C for 2-3 hours at an inoculation amount of 1%. When the OD600 was about 0.5-0.6, IPTG was added for induction (the final concentration of IPTG was 0.1 mM), continued to culture at 37 ° C for 4 hours. The cells were collected by centrifugation, and the supernatant was centrifuged by ultrasonication to determine the soluble expression of recombinant PFA.

The experimental data in Figure 1A shows that the molecular expression of the chaperone protein (whether Prefoldin or Hsp60) and the thermostable amylase PFA can increase the soluble expression level of PFA, especially the effect of Prefoldin. When the chaperone protein Prefoldin is co-expressed with the amylase PFA, After the disruption, the specific activity of PFA in the supernatant was significantly improved compared with the control (PFA), which was more than three times higher than that of the simultaneous expression of the chaperone protein Hsp60.

The results of SDS-PAGE electrophoresis in Fig. 1B also showed that after co-expression of Prefoldin and PFA, the supernatant was centrifuged and the supernatant sample (7 lanes) was compared with other samples and controls (3, 5, 9 lanes), PFA strips. With a significant increase.

The experiment was repeated three times, and the results obtained were consistent, indicating that Prefoldin can significantly increase the soluble expression level of the extreme heat-resistant amylase PFA.

Example 2. Effect of increased expression of Prefoldin on soluble expression of recombinant PFA

In view of the fact that co-expression of the molecular chaperone protein Prefoldin derived from the thermophilic archaea P. furiosus can significantly increase the soluble expression level of the extreme thermostable amylase PFA, the present inventors hope to know whether to further increase the intracellular expression level of the chaperone protein Prefoldin. The soluble expression level of the recombinant extreme thermostable amylase PFA can be further improved.

The expression vector used in the chaperone Prefoldin in Example 1 was pACYC. The inventors cloned the coding sequence of the chaperone protein Prefoldin into the pCDF plasmid and co-transformed with the recombinant amylase PFA expression plasmid into the expression strain. After the strain was taken out from the glycerol tube frozen at -80 ° C, it was inoculated into 3 ml of LB medium having corresponding resistance, and cultured overnight at 37 ° C on a shaker at 250 rpm. The overnight culture medium was transferred to 200 ml of LB medium containing the corresponding resistance at 37 ° C for 2-3 hours at an inoculation amount of 1%. When the OD600 was about 0.5-0.6, IPTG was added for induction (the final concentration of IPTG was 0.1 mM), continued to culture at 37 ° C for 4 hours. The cells were collected by centrifugation, and the supernatant was centrifuged by ultrasonication to determine the soluble expression of recombinant PFA. It was found that the expression level of Prefoldin in pCDF-Prefoldin and recombinant PFA co-expressing strains increased by nearly 2-4 times.

As shown in Fig. 2A, the strain co-expressed with pCDF-Prefoldin and the amylase PFA plasmid was cultured in the disrupted supernatant, and the enzyme activity of PFA was higher than that of the recombinant heat-resistant amylase PFA in the strain co-expressed with pACYC-Prefoldin and PFA. A substantial increase of nearly three times or more. This indicates that the increase in the expression of the chaperone protein Prefoldin further increases the soluble expression level of the recombinant extreme thermostable amylase PFA. The SDS-PAGE results in Figure 2B also showed a significant increase in the soluble expression of the recombinant amylase PFA band after increased Prefoldin expression (lane 8, compared to lane 2 and lane 5).

The above experiment was repeated three times and the results were consistent.

Example 3, co-expression of extreme heat-resistant amylase PFA and Prefoldin

The present inventors further cloned the gene encoding the extreme thermostable amylase PFA gene and the gene encoding the extreme thermophilic archaea P. furiosus chaperone protein Prefoldin in the same plasmid to achieve a common copy number of PFA and Prefoldin gene of 1:1. expression. The thermostable amylase PFA and Prefoldin were co-expressed in both pACYCDuet-1 and pCDFDuet-1 expression vectors, respectively.

The experimental results are shown in Figure 3. The results showed that the expression level of soluble expression of PFA was significantly increased in both pACYC and pCDF vectors when the PFA and Prefoldin were simultaneously expressed in the same plasmid. Simultaneous expression of chaperone and recombinant PFA in the same plasmid enhances the soluble expression level of recombinant PFA, and on the other hand greatly simplifies the construction of strains that facilitate the soluble expression of recombinant PFA, further providing for the future large-scale production of recombinant PFA. Convenience.

in conclusion

Here, a method for significantly increasing the soluble expression of recombinant heat-resistant α-amylase PFA was found. By co-expressing the molecular chaperone protein Prefoldin of the extremely thermophilic archaea P. furiosus in recombinant PFA expressing bacteria, the recombinant extreme heat-resistant α-amylase PFA originally expressed in the form of insoluble inclusion bodies can be more than 50%. The soluble form was expressed (Fig. 2B). It was found in the experiment that Prefoldin derived from the extremely thermophilic archaea P. furiosus can significantly promote the soluble expression of the extreme heat-resistant α-amylase derived from the extremely thermophilic archaea P. furiosus. The method is simple and convenient, and is suitable for large-scale preparation and application of industrialization of extreme heat-resistant α-amylase. It provides strong support for the industrial application of the future recombinant heat-resistant α-amylase. The method has important reference significance for the soluble expression of other recombinant proteins and recombinant industrial enzymes.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

A method for recombinantly expressing an extreme heat-resistant α-amylase, which comprises: co-expressing a gene encoding an extremely thermostable α-amylase with a gene encoding a chaperone protein Prefoldin to obtain a soluble extreme resistance Hot alpha-amylase.
The method according to claim 1, wherein the gene encoding the extreme thermostable alpha-amylase and the gene encoding the chaperone protein Prefoldin are co-expressed in E. coli cells.
The method of claim 2 wherein said method of co-expression comprises:

(1) Providing an expression vector comprising an expression cassette of an extreme heat-resistant α-amylase and an expression cassette of a molecular chaperone protein Prefoldin;

(2) The expression vector of (1) is transformed into Escherichia coli cells, and recombinant E. coli cells transformed with the expression vector are cultured to obtain a soluble extreme heat-resistant α-amylase.
The method according to claim 3, wherein the expression cassette containing the extreme heat-resistant α-amylase and the expression cassette of the molecular chaperone protein Prefoldin are each located in different expression vectors.
The method according to claim 3, wherein the expression cassette containing the extreme heat-resistant α-amylase and the expression cassette of the molecular chaperone protein Prefoldin are located in the same expression vector.
The method of claim 1 wherein said extreme heat-resistant alpha-amylase is an extreme heat-resistant alpha-amylase PFA.
The use of the molecular chaperone protein Prefoldin is used to promote the soluble expression of recombinant heat-resistant alpha-amylase.
The use according to claim 7, wherein the molecular chaperone protein Prefoldin promotes soluble expression of an extremely heat-resistant α-amylase by co-expression with an extremely heat-resistant α-amylase.
Recombinant host cell characterized in that said host cell contains an extremely heat-resistant alpha-starch An expression cassette for the enzyme and an expression cassette for the molecular chaperone protein Prefoldin.
A kit for recombinantly expressing an extreme heat-resistant α-amylase, characterized in that the kit comprises:

An expression vector comprising an expression cassette for an extreme thermostable alpha-amylase and an expression cassette for the chaperone protein Prefoldin.
The kit according to claim 10, wherein said expression vector comprises: pT7473, pACYC or pCDF.
The kit according to claim 10, further comprising: Escherichia coli cells.