CN103074343B

CN103074343B - A kind of method utilizing the endogenous High-expression promoter of 2-DE technology screening Corynebacterium crenatum

Info

Publication number: CN103074343B
Application number: CN201310043507.0A
Authority: CN
Inventors: 饶志明; 许正宏; 张博慧; 徐美娟
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2013-02-04
Filing date: 2013-02-04
Publication date: 2015-10-14
Anticipated expiration: 2033-02-04
Also published as: CN103074343A

Abstract

Utilize a method for the endogenous High-expression promoter of 2-DE technology screening Corynebacterium crenatum, belong to proteomics and genetically engineered field.First the present invention identifies high expression level protein site in Corynebacterium crenatum born of the same parents by 2-DE combine with technique mass-spectrometric technique.Clone its encoding gene promoters, called after P-argC, P-argG, P-argF, P-ilvC and P-serA respectively, replace the tac promotor on shuttle vectors pDXW-8, and insert cat gene in its downstream, transformation of E. coli JM109 and Corynebacterium crenatum SYPA5-5 respectively, by measuring the activity of CAT, the activity of each promotor in recombinant bacterium: P-argG > P-argF > P-ilvC > P-serA > P-argC.This obtains from Corynebacterium crenatum composing type High-expression promoter first, for Corynebacterium crenatum high expression level foreign gene provides effective instrument.

Description

A kind of method utilizing the endogenous High-expression promoter of 2-DE technology screening Corynebacterium crenatum

Technical field

Utilize a method for 2-DE technology screening Corynebacterium crenatum endogenous High-expression promoter, belong to proteomics and genetically engineered field.

Technical background

Bidirectional electrophoresis technique (two-dimensional electrophoresis, 2-DE) is the core technology of proteome research.2-DE technology is independently set up in two laboratories in 1975 respectively by O Farrell and Klose, high-resolution isoelectrofocusing (Iso-electric focusing, IEF) electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) are constituted jointly two-dimensional electrophoresis by them.Its ultimate principle is: first to based on isoelectrofocusing in the pH gradient glue that do not coexist of isoelectric points of proteins; Second to being then separated according to the different of molecular size range along from one to vertical direction, and the protein mixture of complexity is separated on two dimensional surface.

Along with engineered development, usually need the expression vector building a kind of energy high level expression heterologous protein.The expression level impact of promotor on foreign gene is very large, is the critical elements of gene engineering expression carrier.Therefore, study promoter function for understanding biological growth and development, inquire into the mechanism of biological adaptation environment and realize the high expression of foreign gene in genetically modified organism etc. all significant.

Corynebacterium crenatum SYPA5-5 is the mutant strain of a plant height product L-arginine of this seminar screening in early stage, and L-arginine output is up to 36g/L.The structure of its Efficient Genetic expression system plays a crucial role to meta-bolitess such as Corynebacterium crenatum transformation high yield L-arginines.The many employings of current widely used bar bacterium expression vector are from colibacillary promotor as exogenous promoter such as P-tac and lacZ, and these exogenous promoter efficiency are general not high, and its application is by the restriction of many factors.In recent years, bar bacterium internal promoter more and more becomes the focus of bar bacterium genetic expression architectural study.

The present invention utilizes 2-DE technology, and screening high expression level protein site, carries out MALDI-TOF-MS and MS/MS Analysis and Identification and database retrieval to these high expression level protein sites, identify the kind of these protein.According to database retrieval, find out its corresponding coding gene sequence, and clone its promoter sequence.Respectively these promotors are replaced the tac promotor on shuttle vectors pDXW-8, and insert reporter gene chloramphenicol acetyl transferasegene (cat) in its downstream, and difference transformation of E. coli JM109 and Corynebacterium crenatum SYPA5-5, by measuring the activity of CAT, prove that these promotors screened all have certain activity in intestinal bacteria and Corynebacterium crenatum, wherein P-argG, P-argF, P-ilvC activity is higher.

Summary of the invention

The object of the present invention is to provide: screen the method with highly active Corynebacterium crenatum endogenesis promoter by proteomic techniques and genetic engineering means.CAT enzyme activity determination is carried out to the recombinant bacterial strain built and finds that promoter activity is higher, and these promotors are not needing the high expression level that can realize foreign gene under the condition of adding inductor.For Corynebacterium crenatum overexpression foreign gene provides effective instrument.

Technical scheme of the present invention: utilize 2-DE technology, MALDI-TOF-MS and MS/MS Analysis and Identification and database retrieval, filters out the endogenous high expression level protein of Corynebacterium crenatum.Find out its coding gene sequence according to database retrieval, isolate its possible promoter sequence in the upstream of gene order.Pass through molecule clone technology, respectively these promotors are replaced the tac promotor on shuttle vectors pDXW-8, and insert reporter gene chloramphenicol acetyl transferasegene (cat) in its downstream, and difference transformation of E. coli JM109 and Corynebacterium crenatum, by measuring the activity of CAT, the activity of checking promotor in Host Strains.

(1) the endogenous high activity promoter of bar bacterium is screened according to 2-DE:

1., after yeast culture actication of culture, be in the Erlenmeyer flask of 50mL/250mL by 5% inoculum size access liquid amount, at 30 DEG C, 200rpm (reciprocal shaker) ferments 36h.

2. protein sample is prepared and quantitatively medium centrifugal is collected thalline, and ultrapure water cleans 3 times, cleans 2-3 all over supreme clearly limpid with TE damping fluid (pH7.5); With the resuspended thalline of 20mL TE damping fluid, add 0.2mL PMSF solution (10%100mM proteinase inhibitor), ultrasonic disruption 30min (broken 5s under condition of ice bath.Interval 5s); Add rapidly the RNaseA of DNase I and 20U of 100U, at 37 DEG C, hatch 30min; Centrifuging and taking supernatant adds the ice acetone of precooling, precipitates overnight at-20 DEG C; Centrifugal collecting precipitation, it is resuspended to add lysate (8M urea, 2M thiocarbamide, 4%CHAPS, 0.5% amphotericeledrolyte, 1%DTT, 0.001% tetrabromophenol sulfonphthalein).Bradford method quantification of protein ,-70 DEG C save backup.

3. two-dimensional electrophoresis every clotting glue loading 400 μ g albumen, mix with hydrating fluid (8mM urea, 2mM thiocarbamide, 4%CHAPS, 1%DTT, 0.5% amphotericeledrolyte and 0.001% tetrabromophenol sulfonphthalein), loading volume is 450 μ L.By IPG adhesive tape (17cm, pH4-7) rehydration, cover mineral oil.Isoelectrofocusing program is 500V2h → 1000V3h → 10000V4h → 10000V40000Vh → 500V10h.After isoelectrofocusing terminates, adhesive tape is placed in balance liquid (6M urea, 2%SDS, 0.375pH8.8This-HCl, 20% glycerine) and balances 2 times, and vibrate 10-15min at every turn.Wherein add 20mM DTT in flat liquid weighing apparatus I, in balance liquid II, add 100mM iodo-acid amide.After having balanced, adhesive tape level is placed in ready made 12%SDS gel top, with the liquid-solid fixed bubble removing side by side of agarose sealing, carries out the 2nd to gel electrophoresis.Often kind of cell protein sample repeats 3 plate glue.Constant current 32mA30min, 48mA5h40min, until tetrabromophenol sulfonphthalein forward position arrives bottom sheet glass.With 0.03% coomassie brilliant blue R_250 dyeing 2h, spend the night with 10% acetic acid decolouring.

4. gel image analysis and protein site mass fingerprint analysis Lad Scan (GE Health) scan gel, then utilize ImageMaster2D Platinum5.0 software to adjust gel images, look for point, quantitatively and right.Extract protein site supernatant Zip TipC18 (millipore) the microtrabeculae desalination after decolouring, drying and trypsin digestion chosen.Sample is placed on Bruker Autoflex MALDI-TOF-MS mass spectrograph and carries out mass spectroscopy, obtains peptide fingerprinting spectrum.For determining further, choosing ten the highest peak values and carrying out MS/MS analysis.

(2) construction process of recombinant expression vector and bacterial strain:

1. the structure of recombinant expression vector pDXW-8-cat is according to the sequence of chloramphenicol acetyl-transferase gene (cat) in carrier pACYCDuet-1, and the restriction enzyme site design cat gene primer on pDXW-8 plasmid is:

F：5’-ACCCG GAATTCGAAAGGACATGAACGATGGAGAAAAAAATCACTGG-3’；

R：5’-CGC AAGCTTTCATTCTTCGTCACCTCC-3’

Take pACYCDuet-1 as template, carry out the gene order that pcr amplification goes out cat.Reclaim test kit specification sheets with reference to vast Imtech glue and reclaim PCR primer, glue recovery product spends the night with pMD18-T by a certain percentage and is connected, Transformed E .coliJM109 competent cell, use amicillin resistance plate screening recombinant bacterium, recombinant plasmid through PstI/NdeI digestion verification, recombinant plasmid called after pMD18-T-cat.Extract plasmid pMD18-T-cat and pDXW-8 be stored in E.coli JM109, two plasmids are respectively through Pst I/ Nde I double digestion, and glue reclaims purifying, T ₄dNA ligase spends the night connection two fragment, by connector thermal shock Transformed E .coli JM109 competent cell after spending the night, uses kalamycin resistance plate screening positive transformant.Extract transformant plasmid, recombinant plasmid is verified through Pst I/NdeI double digestion, recombinant plasmid called after pDXW-8-cat.

2. the preparation of several promoter dna fragment is according to two dimensional electrophoresis qualification result, GENBANK website is found their gene order, according to these sequences Design synthetic promoter oligonucleotide single stranded sequences, and introduce EcoR I and BamH I restriction enzyme site in the sequence, with Corynebacterium crenatum SYPA5-5 chromosomal DNA for template amplification goes out each promoter sequence, respectively called after P-argC, P-argG, P-argF, P-ilvC, P-serA.

Structure and the conversion of 3. carrying the recombinant promoter carrier detection of promoter fragment carry out purifying with reference to vast Tyke isogeneity test kit to above-mentioned promoter fragment PCR primer, purified product and pDXW-8-cat use EcoR I/BamH I enzyme to cut, after glue reclaims purifying, T4DNA ligase enzyme spends the night connection, connect product thermal shock Transformed E .coli JM109 competent cell, electroporated method transforms Corynebacterium crenatum SYPA5-5.Use kalamycin resistance plate screening positive transformant.Extract transformant plasmid, recombinant plasmid is verified through PCR, and recombinant plasmid is called after pDXW-P respectively _argC-cat, pDXW-P _argG-cat, pDXW-P _argF-cat, pDXW-P _ilvC-cat and pDXW-P _serA-cat.

(3) the mensuration recombination bacillus coli JM109 that recombinant bacterium CAT enzyme is lived is inoculated in respectively in LB and LBG substratum with restructuring bar bacterium and cultivates, collected by centrifugation thalline, thalline is washed twice with 100mM Tris-HCl (pH7.8), ultrasonic disruption is carried out after the Tris-HCl of certain volume is resuspended, broken rear 10,000rpm is centrifugal, and 30min gets supernatant, i.e. crude enzyme liquid.CAT enzyme activity determination method: containing 100mM Tris-HCl (pH7.8), 0.1mM acetyl-CoA, 0.4mg/ml DTNB in reaction system, appropriate crude enzyme liquid; Reaction mixture is heated to 37 DEG C in a water bath, adds paraxin to final concentration 0.1mM, mixing, measure absorbance value A immediately ₄₁₂, not add the reaction solution of paraxin for contrast.A unit of activity is defined as: the enzyme amount needed for per minute acetylize 1 μm of ol paraxin.

Beneficial effect of the present invention: the present invention utilizes 2-DE combine with technique Mass Spectrometric Identification technology screening to go out Corynebacterium crenatum 5 high expression level protein sites.Its coding gene sequence is found out by database retrieval, clone their promotor and the tac promotor of replacing on shuttle vectors pDXW-8, and insert reporter gene chloramphenicol acetyl transferasegene (cat) in its downstream, transformation of E. coli JM109 and Corynebacterium crenatum respectively, by measuring the activity of CAT, prove that these promotors screened all have certain activity in intestinal bacteria and Corynebacterium crenatum, wherein P-argG, P-argF, P-ilvC activity is higher.

These promotors can realize the high expression level of foreign gene under the condition not needing interpolation inductor, for Corynebacterium crenatum overexpression foreign gene provides effective instrument.

Accompanying drawing explanation

Fig. 1 Corynebacterium crenatum soluble proteins sample two-dimensional electrophoresis gel images

Fig. 2 pDXW-8-cat proof diagram

1λDNA/HindIII markers

2pDXW-8-cat/PCR

3pDXW-8-cat/EcoR I

4pDXW-8-cat/EcoR I+BamH I

5pDXW-8/EcoR I+BamH I

6DNA Marker DL2000

Fig. 3 recombinates the mensuration of E.coli JM109CAT enzyme activity

The mensuration of Fig. 4 recombinant C .crenatum SYPA5-5CAT enzyme activity

Specific implementation method

Embodiment 1: screen the endogenous high activity promoter of Corynebacterium crenatum according to 2-DE

Thalline is through cultivation, proteins extraction, and the protein concn extracted is about 70 μ g/ μ L, and first to isoelectrofocusing, balance and second to SDS-PAGE, through fixing, dye and after decolouring, obtaining protein distribution gel clearly; With Lad Scan (GE Health), gel is scanned, then utilize ImageMaster 2D Platinum5.0 software to adjust gel images, look for point, quantitatively and right, extract the protein site chosen and carry out MALDI-TOF-MS and MS/MS analysis, successful identification goes out 15 high expression level protein sites altogether.

Embodiment 2: the structure of recombinant expression vector and bacterial strain

Based on 15 the high expression level protein sites obtained in embodiment 1, in conjunction with information such as its copy numbers in somatic cells, have found 5 target protein points, the gene order that this 5 kinds of protein are corresponding is found in GENBANK, and be separated its promoter sequence according to gene order, respectively by these 5 promotor called afters P-argC, P-argG, P-argF, P-ilvC and P-serA.Respectively these 5 promotors are replaced the tac promotor on shuttle vectors pDXW-8, and insert reporter gene chloramphenicol acetyl transferasegene cat gene in its downstream, recombinant vectors is transformed in e. coli jm109 and Corynebacterium crenatum.Through checking, successfully construct pDXW-P _argC-cat, pDXW-P _argG-cat, pDXW-P _argF-cat, pDXW-P _ilvC-cat and pDXW-P _serA-cat, and the recombinant bacterium successfully obtaining its correspondence.

Embodiment 3: the enzyme activity determination of recombinant bacterial strain

Recombinant bacterial strain is cultivated respectively in LB and LBG substratum, the 100mMTris-HCl buffer solution for cleaning of 8000rpm centrifugal 10min, pH7.8 2 times, and be suspended in this damping fluid, crude enzyme liquid is prepared in ultrasonic disruption process.Reaction mixture is made up of the DTNB of the crude enzyme liquid of certain volume, the Tris-HCL (PH7.8) of 100mM, acetyl-CoA, 0.4mg/mL of 0.1mM, 37 DEG C of reaction 2min, add paraxin termination reaction, detect the light absorption value changing conditions under 412nm.Enzyme lives unit definition for enzyme amount needed for per minute acetylize 1 μm of ol paraxin.

Enzyme activity such as the Fig. 3 recording recombination bacillus coli is respectively: E.coli JM109/pDXW-P _argC-cat is 0.36U/mg, E.coli JM109/pDXW-P _argG-cat is 5.58U/mg, E.coli JM109/pDXW-P _argF-cat is 3.81U/mg, E.coli JM109/pDXW-P _ilvC-cat is 3.73U/mg, E.coli JM109/pDXW-P _serA-cat is 1.26U/mg.

Enzyme activity such as the Fig. 4 recording recombinant corynebacterium crematum is respectively: C.crenatum SYPA5-5/pDXW-P _argC-cat is 0.09U/mg, C. crenatum SYPA5-5/pDXW-P _argG-cat is 1.86U/mg, C.crenatum SYPA5-5/pDXW-P _argF-cat is 1.19U/mg, C.crenatum SYPA5-5/pDXW-P _ilvC-cat is 1.12U/mg, C.crenatumSYPA5-5/pDXW-P _serA-cat is 0.089U/mg.

Above result can find out that P-argG, P-argF, P-ilvC CAT enzyme activity in E.coli JM109 and C.crenatum SYPA5-5 is all higher, illustrates that it has higher vigor.This obtains first to come from composing type High-expression promoter in Corynebacterium crenatum, for Corynebacterium crenatum high expression level foreign gene provides effective instrument.

Claims

1.N-acetyl-gamma-glutamyl amine phosphate dehydrogenase gene promoter, is characterized in that: sequence is as shown in SEQ ID NO:1.