CN109385440A

CN109385440A - A method of destination protein expression in regulation bacillus subtilis

Info

Publication number: CN109385440A
Application number: CN201811241708.0A
Authority: CN
Inventors: 刘延峰; 堵国成; 田荣臻; 陈坚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-10-24
Filing date: 2018-10-24
Publication date: 2019-02-26

Abstract

The invention discloses the methods and application of destination protein expression in a kind of regulation bacillus subtilis, belong to gene engineering technology field.The present invention is using bacillus subtilis (Bacillus subtilis) as expressive host, by adding the amino acid sequence designed by ad hoc approach before destination protein N-terminal encoding gene, and the expression that dissociates in plasmid, recombined bacillus subtilis destination protein expression quantity is set to improve 397 times to transformation, 1.94% before being reduced to transformation.This lays a good foundation for Bacillus subtilis genes albumen high efficient expression and genetic engineering transformation.

Description

A method of destination protein expression in regulation bacillus subtilis

Technical field

The present invention relates to the methods and application of destination protein expression in a kind of regulation bacillus subtilis, belong to genetic engineering Technical field.

Background technique

Model microorganism of the bacillus subtilis (Bacillus subtilis) as gram-positive bacteria is used for spore The laboratory research of Forming Mechanism and Metabolism regulation, it is widely distributed in nature, including soil surface, water environment and animal stomach. It is a kind of non-pathogenic microorganism, is free of endotoxin, and be usually recognized as safety by food and drug administration (FDA) (GRAS) food-grade microorganisms.In addition, bacillus subtilis has many advantages, comprising: cell growth is fast, and incubation time is short, Fostering requirement is low, and secretory protein ability is strong, is widely used in industrial enzyme production, the biosynthesis of high value added product.However, adjusting The building in control element library still lags behind other important model organisms, for example, Escherichia coli (Escherichia coli) and Saccharomyces cerevisiae (Saccharomyces cerevisiae).

In order to adjust the gene expression in bacillus subtilis, the standard Expression element for the bacillus subtilis applied at present It mainly include promoter, terminator, RBS sequence, SPACER etc..In addition, having had been built up and subtilisin matter table Up to relevant more and more Genetic Toolbox.However, current Expression element is respective using having the shortcomings that, including effect is not Obviously, operating cost height etc..In addition, most standard element lays particular emphasis on the adjusting of transcriptional level, translation skill control tool and Component is less, and translation skill also has a significant impact to gene expression.Therefore, it is developed in bacillus subtilis wieldy Expression regulation element is particularly significant.

Summary of the invention

It is described the first purpose of the invention is to provide a kind of method of destination protein expression in regulation bacillus subtilis Method is transformed to the N-terminal sequence of destination protein, and improved destination protein is transferred to carry out table in bacillus subtilis Reach, the N-terminal sequential element is the coded sequence of destination protein the 1-15 amino acid of N-terminal, the method for the transformation include with Descend any one or a few:

(1) by destination protein N-terminal sequence serine (S), tryptophan (W), aspartic acid (D), cysteine (C), Glycine (G), leucine (L), histidine (H), arginine (R) are classified as the first candidate locus；When the 2nd non-asparagine of N-terminal (N), lysine (K) or when glutamic acid (E) ranks N-terminal the 2nd for the second candidate locus, then by the first candidate locus, Two candidate locus replace with asparagine, lysine, any one in glutamic acid；When replacement, meet following (a) and (b):

(a) when the first candidate locus, the previous amino acid of the second candidate locus or the latter amino acid be asparagine, When any one amino acid in lysine, glutamic acid, which is substituted for and its previous amino acid or the latter The identical amino acid of amino acid, still, if being wrapped from 7 amino acid of the front three to latter three of the candidate locus after replacement Continuous 4 or more the amino acid being made of any one or more in asparagine, lysine, glutamic acid is contained, then will The candidate locus replaces with glutamine (Q) or isoleucine (I)；

(b) it under the premise of preferentially meeting (a), is substituted for the quantity of the candidate locus of asparagine and is substituted for lysine The quantity of candidate locus, the difference of any one in the quantity for the candidate locus for being substituted for glutamic acid be no more than 2；

(2) asparagine in destination protein N-terminal sequence, lysine, glutamic acid, site where glutamine are classified as the Then third candidate locus is successively substituted for serine, tryptophan, aspartic acid, cysteine by three candidate locus in order.

In one embodiment of the invention, replaced asparagine uses codon AAC or AAT, bad ammonia in (1) Acid uses codon CAA using codon GAA, glutamine using codon AAA, glutamic acid.

In one embodiment of the invention, replaced serine uses codon TCC or TCT, tryptophan in (2) Codon TGT is used using codon GAT, cysteine using codon TGG, aspartic acid.

In one embodiment of the invention, (a) (b) (c) belongs to the N-terminal sequence for improving subtilisin expression Column element remodeling method (d) belongs to the N-terminal sequential element remodeling method for reducing subtilisin expression.

In one embodiment of the invention, the bacillus subtilis includes Bacillus subtilis 168.

In one embodiment of the invention, the destination protein includes zymoprotein, non-zymoprotein.

Second object of the present invention is to improve a kind of recombined bacillus subtilis, with Bacillus subtilis 168 Above-mentioned improved destination protein is expressed using pP43NMK as expression vector for host.

Third object of the present invention is to provide above-mentioned recombined bacillus subtilis to produce answering in destination protein in fermentation With.

The present invention provides a kind of N-terminal sequential element design methods of promotion subtilisin expression, with fluorescence Intensity characterizes expressing quantity according to fluorescent strength determining method described herein Example obtains SEQ ID NO.26, when fermentation time is 4h hours or so, relative intensity of fluorescence is mentioned from 55 after artificial reconstructed Up to 21948.3, rise to original 397 times；By taking original N-terminal sequence SEQ ID NO.3 as an example, after artificial reconstructed, obtain To SEQ ID NO.31, when fermentation time is 4h hours or so, relative intensity of fluorescence is reduced to 432.9 from 22320.6, is reduced to Originally 1.94%.

Specific embodiment

(1) recombined bacillus subtilis seed culture and fermentation

Culture medium (g/L): tryptone 10, yeast powder 5, NaCl 10.

Condition of culture: being transferred to fermentation medium for the seed for cultivating 10h under 37 DEG C, 200rpm with 10% inoculum concentration, in 37 DEG C, cultivate 20h under the conditions of 200rpm.

(2) measuring method of egfp expression amount

The fermentation liquid after 200 μ L dilution is added in every hole in 96 orifice plates, is detected using Cytation3 cell imaging microwell plate Instrument (Bai Teng Instrument Ltd., the U.S.), excitation wavelength: 488nm, launch wavelength: 523nm, gain: 60.

The building of 1 recombinant plasmid of embodiment

The composition of recombinant plasmid is to be sequentially inserted into coding N-terminal in order after the P43 promoter of Pp43NMK plasmid Sequence and green fluorescent protein (GFP) gene.

After sequence introducing P43 promoter in order to which N-terminal will be encoded, design primer rh_Inc1-0.75k_p43NMK- GFP_F: sequence information is as shown in SEQ ID NO.36；Rh_Inc1-0.75k_p43NMK-GFP_R: sequence information such as SEQ ID Shown in NO.37.Using plasmid pP43NMK as template, the N section that transformation is inserted after obtaining P43 promoter is reversely expanded by PCR The plasmid fragments of sequence.

With the Escherichia coli containing green fluorescent protein (GFP, GenBank:AF324408.1) gene for template, design is drawn Object obtains gfp fragment by bacterium colony PCR；Primer is as follows:

Fx_Inc1-6.7k_p43NMK-GFP_F: sequence information is as shown in SEQ ID NO.38；

Fx_Inc1-6.7k_p43NMK-GFP_R: sequence information is as shown in SEQ ID NO.39.

Change finally, will be inserted by Gibson Assembly Clonging Kit (New England Biolabs) The plasmid fragments for the N section sequence made and the genetic fragment of encoding green fluorescent protein recombinate, and building obtains recombinant plasmid, and sequencing is tested Card, confirmation recombination pP43NMK-Design1-GFP plasmid construction success.

The building of the recombination pP43NMK-Design1-GFP plasmid bacillus subtilis of embodiment 2

The pP43NMK-GLNA-GFP plasmid built is transformed into 168 wild-type strain of bacillus subtilis.Using yz_ Zong-p43NMK_F: sequence information is as shown in SEQ ID NO.40 and yz_zong-p43NMK_R: sequence information such as SEQ ID Shown in NO.41, primer selects transformant and carries out bacterium colony PCR, carries out SDS-PAGE, 1.8kb band occurs, verifies recombinant bacillus bud Spore bacillus constructs successfully.

Embodiment 3 adds the influence that engineer's N-terminal sequence expresses green fluorescent protein in engineering bacteria

The recombined bacillus subtilis seed of 9h will be cultivated in 37 DEG C, 750rpm, 700 μ L LB culture mediums, 96 hole deep-well plates Liquid is transferred to 190 μ L LB culture mediums with 5% inoculum concentration, and 4h is cultivated under the conditions of 37 DEG C, 750rpm and obtains recombinant bacillus gemma bar Bacteria culture fluid sample.Under the same terms, the final fermentation broth sample of recombined bacillus subtilis sample of original N-terminal sequence is added In measure 4 hourly average value of fluorescence intensity be 32608；And pass through after artificial reconstructed N-terminal sequence, measure recombinant bacillus gemma bar 4 hourly average value of bacteria culture fluid fluorescent intensity is 42986.

The original N-terminal sequence of 14 kinds of differences is shown in Table 1 for the impact effect of egfp expression amount after artificial reconstructed.

14 N-terminal sequences, which are transformed, in table 1 influences situation to protein expression

Reference examples 1 are constructed without specific N-terminal sequence control group

The composition for recombinating control plasmid is to be inserted directly into green fluorescent protein after the P43 promoter of Pp43NMK plasmid (GFP) gene.

Design primer rh_Ctr-0.75k_p43NMK-GFP_F: sequence information is as shown in SEQ ID NO.42, rh_Ctr- 0.75k_p43NMK-GFP_R: sequence information as shown in SEQ ID NO.43, with containing green fluorescent protein (GFP, GenBank: AF324408.1) Escherichia coli of gene obtain gfp fragment by bacterium colony PCR for template；Design primer fx_ Ctr-6.7k_p43NMK-GFP_F: sequence information is as shown in SEQ ID NO.44；Fx_Ctr-6.7k_p43NMK-GFP_R: sequence Column information is as shown in SEQ ID NO.45.Using plasmid pP43NMK as template, is reversely expanded by PCR and obtain plasmid fragments；Finally By Gibson Assembly Clonging Kit (New England Biolabs) construction recombination plasmid, sequence verification, really After recognizing recombination pP43NMK-Ctr-GFP plasmid construction success, 168 wild type of bacillus subtilis is converted.Bacterium colony PCR verifies plasmid It converts successfully.

By under 37 DEG C, 200rpm cultivate 10h seed fermentation medium is transferred to 10% inoculum concentration, in 37 DEG C, 20h is cultivated under the conditions of 200rpm.Measuring fluorescence intensity in final 4h fermentation liquid is 5159, the control as each fermentation verifying.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

<110>Southern Yangtze University

<120>a kind of method that destination protein is expressed in regulation bacillus subtilis

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<170> PatentIn version 3.3

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acacatgagt aaaggagaag aacttttcac tggagttgtc cca 43

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cttctccttt actcatgtgt acattcctct cttacctata atggtaccgc tatcactt 58

Claims

1. a kind of method of destination protein expression in regulation bacillus subtilis, which is characterized in that the N-terminal sequence of destination protein It is transformed, improved destination protein is transferred in bacillus subtilis and is expressed, the N-terminal sequential element is purpose egg The coded sequence of white the 1-15 amino acid of N-terminal, the method for the transformation include any of the following or several:

(1) by serine (S), tryptophan (W), aspartic acid (D), the cysteine (C), sweet ammonia in destination protein N-terminal sequence Sour (G), leucine (L), histidine (H), arginine (R) are classified as the first candidate locus；When N-terminal the 2nd non-asparagine (N), N-terminal the 2nd is ranked for the second candidate locus, then waits the first candidate locus, second by lysine (K) or when glutamic acid (E) Bit selecting point replaces with asparagine, lysine, any one in glutamic acid；When replacement, meet following (a) and (b):

(a) when the first candidate locus, the previous amino acid of the second candidate locus or the latter amino acid are asparagines, rely ammonia When acid, any one amino acid in glutamic acid, which is substituted for and its previous amino acid or the latter amino The identical amino acid of acid, still, if being contained from 7 amino acid of the front three to latter three of the candidate locus after replacement Continuous 4 or more the amino acid being made of any one or more in asparagine, lysine, glutamic acid, then by the time Bit selecting point replaces with glutamine (Q) or isoleucine (I)；

(b) it under the premise of preferentially meeting (a), is substituted for the quantity of the candidate locus of asparagine and is substituted for the time of lysine The difference of any one in the quantity of bit selecting point, the quantity for the candidate locus for being substituted for glutamic acid is no more than 2；

(2) asparagine in destination protein N-terminal sequence, lysine, glutamic acid, glutamine place site are classified as third and waited Bit selecting point, is then successively substituted for serine, tryptophan, aspartic acid, cysteine for third candidate locus in order.

2. the method according to claim 1, wherein in (1) replaced asparagine using codon AAC or AAT, lysine use codon CAA using codon GAA, glutamine using codon AAA, glutamic acid.

3. the method according to claim 1, wherein in (2) replaced serine using codon TCC or TCT, tryptophan use codon TGT using codon GAT, cysteine using codon TGG, aspartic acid.

4. method according to claim 1 or 2, which is characterized in that (1) belong to and improve subtilisin expression N-terminal sequential element remodeling method.

5. method according to claim 1 or 3, which is characterized in that (2) belonging to reduces subtilisin expression N-terminal sequential element remodeling method.

6. the method according to claim 1, wherein the N-terminal sequence of the improved destination protein includes SEQ NO.15~35 ID.

7. -6 any method according to claim 1, which is characterized in that the bacillus subtilis includes Bacillus subtilis168。

8. method according to claim 1 or claim 7, which is characterized in that the destination protein includes zymoprotein, non-zymoprotein.

9. a kind of recombined bacillus subtilis, which is characterized in that with Bacillus subtilis 168 be host, with pP43NMK For expression vector, improved destination protein described in claim 6 is expressed.

10. recombined bacillus subtilis as claimed in claim 9 produces the application in destination protein in fermentation.