CN102021195B - Method for efficiently preparing protein molecular weight standard by utilizing prokaryotic expression system - Google Patents
Method for efficiently preparing protein molecular weight standard by utilizing prokaryotic expression system Download PDFInfo
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- CN102021195B CN102021195B CN2010105173530A CN201010517353A CN102021195B CN 102021195 B CN102021195 B CN 102021195B CN 2010105173530 A CN2010105173530 A CN 2010105173530A CN 201010517353 A CN201010517353 A CN 201010517353A CN 102021195 B CN102021195 B CN 102021195B
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Abstract
The invention discloses a method for efficiently preparing a protein molecular weight standard by utilizing a prokaryotic expression system, which comprises the following steps of: (1) selecting a common low molecular weight standard as an initiation protein molecular weight standard A; (2) selecting a gene sequence with low codon content or without a rare codon as a template, and designing a primer to sequentially obtain different target fragments, so that the molecular weight of an encoding area after a target sequence and a pET28a vector are connected is the set molecular weight in (1), and the optimum number of isoelectric points is 4-9; (3) respectively inserting the target fragments into an expression vector pET28a-(+) according to certain enzyme cutting sites, and respectively transferring screened positive clones into a colon bacillus BL21_star(DE3) for induced expression; (4) verifying the expression forms of different proteins; and (5) respectively purifying target protein by the methods of affinitive layer purification, purification of inclusion bodies and the like, i.e. the protein molecular weight standard.
Description
Technical field
The present invention relates to a kind of method of utilizing prokaryotic expression system efficient production protein molecular weight standard, can be applicable to fields such as biotechnology and biotechnology research.
Background technology
Protein research is an important component part in the life science, and wherein protein molecular weight standard can be used as the important instrument of the qualitative even quantitative examination of protein, can be used for protein electrophorese; Experiments such as the quantification of protein and the protein immunization marking, at present, the protein molecular weight standard source is narrower; Mostly numerous protein molecular weight standards is known native protein; Separation and purification is complicated, and values for molecular weight differs greatly, and because the complicacy that natural protein is modified; The indefinite problem of indication appears in the difference that the same electrophoretic mobility of protein of different sources often occurs.These standard molecular weight albumen usually use the mode of dying in advance in western hybridization simultaneously, and dyestuff arrives electrophoretic mobility and indicating effect with the differentia influence that combines firmness and binding capacity of protein molecular weight standard, costs an arm and a leg simultaneously.Maturation along with protein allos recombination and expression techniques; In order to make purifying recombinant proteins more convenient; Increasing recombinant expression protein end carries the label that is beneficial to affinity purification such as his-tag etc.; These proteic electrophoresis detection usually depend on SDS-PAGE and western hybridization technique, and often there are above-mentioned problems in the molecular weight of albumen standard of in analytic process, using, and the molecular weight of albumen standard can not be shown in the target protein testing process synchronously simultaneously; Cause practical problemss such as operating process is numerous and diverse, therefore exploitation has significant values with the protein molecular weight standard that target protein shows synchronously.
The invention provides High-efficient Production and carry the technology of specific label protein molecular weight standard series, label is given the characteristic that standard molecular weight albumen is easy to detect indication.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art, and a kind of method of utilizing prokaryotic expression system efficient production protein molecular weight standard is provided.
The present invention adopts following technical scheme:
A kind of method of utilizing prokaryotic expression system to prepare protein molecular weight standard may further comprise the steps:
(1) selecting lower molecular weight standard commonly used is initiation protein molecular weight standard A, and middle molecular weight of albumen is pressed A+15, and A+30, A+45, A+60, A+75, terminal point albumen press the A+105 molecular weight standard and set;
(2) be chosen in the intestinal bacteria system and do not have or the gene order of a small amount of rare codon is that template design primer is obtained the various objectives fragment successively; Obtain the sequence of truncated gene through the online software analysis of Expasy tools; Aim sequence is connected the molecular weight of coding region, back for setting molecular weight in (1) with the pET28a carrier, the righttest iso-electric point is between 4-9;
(3) the purpose fragment is inserted expression vector pET28a respectively according to certain restriction enzyme site, the positive colony of screening changes e. coli bl21 star (DE3) respectively over to and carries out abduction delivering;
The engineering bacteria of expressing a plurality of molecular weight respectively that (4) will build carries out the optimization of induction time and inductor final concentration respectively;
(5) different albumen are carried out the evaluation of expression-form;
(6) according to the difference of the expression-form of target protein respectively with method purifying target proteins such as affinitive layer purification and inclusion body purifications, be protein molecular weight standard;
The described method of utilizing prokaryotic expression system to prepare protein molecular weight standard; The protein molecular weight standard that step (1) is set increases progressively by 15 or 30 kilodalton gradients from initial albumen; At interval evenly; The gradient that wherein increases progressively between lower molecular weight is 15kD, and the gradient that increases progressively between HMW is 30kD.
The described method of utilizing prokaryotic expression system to prepare protein molecular weight standard, step (2) obtains target gene fragment through the method that designs primer and pcr amplification respectively; Utilize the ATG of carrier simultaneously, final expressed proteins N end has the his label.
Described prokaryotic expression system prepares the method for protein molecular weight standard; After step (3) comprises that also the different purpose gene that step (2) is obtained inserts expression vector pET-28a+ according to certain restriction enzyme site; Change e. coli bl21 (DE3)-star over to, obtain the engineering bacteria of a series of marking protein molecular weight standards.
Described prokaryotic expression system prepares the method for protein molecular weight standard, and step (4) comprises also that the engineering bacteria with described a series of marking protein molecular weight standards is inoculated in respectively and contains 50 μ g/ml cards and receive in the LB liquid nutrient medium of mycin 220rpm/min; 37 ℃ of overnight cultures; Ratio enlarged culturing 3h after added sec.-propyl-β-D-galactoside in 1: 50 next day is 1.0mmol/L to final concentration, respectively at inducing 3h, 5h; 7h; Get with volume appearance, 12000rmp/min centrifugal 4min, deposition thalline behind 9h and the 11h; Resuspended with the 1*SDS-PAGE sample-loading buffer, boiling water boils 5-10min centrifuging and taking supernatant and analyzes in SDS-PAGE, confirms optimum induction time.
Described prokaryotic expression system prepares the method for protein molecular weight standard; Step (4) also comprise with the engineering bacteria of a series of marking protein molecular weight standards be inoculated in 220rpm/min in the LB liquid nutrient medium that contains 50 μ g/ml kantlex respectively, 37 ℃ of overnight cultures added sec.-propyl-β-D-galactoside in 1: 50 and be respectively 0.2 to final concentration next day behind the ratio enlarged culturing 3h; 0.5; 0.8 1.1mmol/L gets after inducing 5h with volume appearance, 12000rmp/min simultaneously; Centrifugal 4min, the deposition thalline; Resuspended with the 1*SDS-PAGE sample-loading buffer, boiling water boils 5-10min centrifuging and taking supernatant and analyzes in SDS-PAGE, confirms the righttest inductor final concentration.
Described prokaryotic expression system prepares the method for protein molecular weight standard, and step (5) is with described optimum induction time and the righttest inductor final concentration, and the optimum that is applied to engineering bacteria is induced; It is resuspended with phosphoric acid buffer and 1%TritonX-100 to collect bacterial sediment; Ice bath, carrying out ultrasonic bacteria breaking 10min, the high speed frozen centrifugation is also collected respectively and is gone up cleer and peaceful deposition; Carry out SDS-PAGE in a small amount and analyze, identify proteic expression-form.
Described prokaryotic expression system prepares the method for protein molecular weight standard; Step (6) combines the nickel post with the said albumen supernatant that is accredited as soluble form that obtains; Earlier wash foreign protein with PBS, using concentration again is to contain the PBS wash-out of 150mmol/L imidazoles and collect target protein; Its deposition of albumen that is accredited as the inclusion body form is earlier with twice of NaCl solution washing; Then with phosphoric acid buffer PBS and the resuspended carrying out ultrasonic bacteria breaking 2min of 1%TritonX-100; Use inclusion body washings washed twice again, use inclusion body washings carrying out ultrasonic bacteria breaking 2min then, at last with inclusion body washings washing three times; Obtain target protein, be protein molecular weight standard.
Described prokaryotic expression system prepares the method for protein molecular weight standard; The recombinant protein label that utilizes prokaryotic expression system to express comprises the antibodies district of His-tag, GST tag, MBP tag, Strep tag and staphylococcus aureus protein A; The corresponding protein molecular weight standard that produces can show in testing process simultaneously with the albumen to be detected with label of the same race, can indicate proteic molecular weight to be detected and content.
The present invention has chosen in the intestinal bacteria system not to be had or the gene order of a small amount of rare codon is that template design primer is obtained the purpose fragment and is connected the intestinal bacteria system that changes over to pET-28a (+) fragment; Thereby having overcome in the expression process because of rare codon causes expression to interrupt or the few shortcoming of expression amount; Realized expressing fast and efficiently of protein molecular weight standard; Utilize the ATG of carrier in expressing simultaneously; Make each proteic N end all have 6*his, developed and got final product the indicator protein polyacrylamide gel electrophoresis, indicate the target protein molecular weight; Can indicate other protein molecular weight standards again, for protein research provides an important reagent with label protein.
Compared with prior art; The present invention utilizes the prokaryotic expression system growth and breeding fast; Advantages such as the level height of simple to operate, cheap, exogenous gene expression product and amalgamation and expression are artificially set protein molecular weight standard commonly used; Utilize the protein molecular weight standard that the prokaryotic expression system efficient production is even at interval, special tag is arranged, the standard molecular weight albumen of formation has the notable feature of being convenient to detect indication.
Description of drawings
Fig. 1 is target gene PCR amplification figure;
Fig. 2 is recombinant plasmid double digestion figure;
Fig. 3 target protein abduction delivering figure;
Fig. 4 detects his label figure behind the target protein purifying.
Embodiment
Below in conjunction with specific embodiment and accompanying drawing the present invention is explained further details, said experimental example is intended to specifically illustrate the present invention with way of example.The selection of label, templet gene, reagent, the value of temperature and its dependent variable and carrier and host's selection just illustrates application of the present invention, and is not construed as limiting the invention, and below explanation is not used in restriction scope of the present invention.
Selecting of 1 molecular weight standard
According to the principle of widespread popularity, selecting 15kD is initial molecular weight of albumen standard, interrupts albumen and is followed successively by 30kD, 45kD; 60kD, 75kD, 90kD; And terminal point protein 12 0kD, the gradient that increases progressively between lower molecular weight is 15kD, the gradient that increases progressively between HMW is 30kD.
2 primer design
According to barley CesA full-length gene and intestinal bacteria lacZ and DNA ligase gene order is template; Application software primer 5 design primers; And the upstream and downstream primer 5 ' end is introduced restriction enzyme site, guarantees that the expression product of last total coding sequence is the standard molecular weight of above design:
The 3PCR amplifying target genes
(1) PCR reaction amplifying target genes: respectively with BL21 (DE3) lacZ gene, barley cesA and BL21 (DE3) DNA ligase gene is an amplification template, and primer separately is according to following PCR reaction system amplification purpose fragment
10×pfu?buffer 2.5μl
10mM?dNTP 0.5μl
10 μ M upstream primers, 0.5 μ l
10 μ M downstream primers, 0.5 μ l
Template (50ng) 1 μ l
ddH
2O 19.5μl
pfu?turbo 0.5μl
25μl
(2) PCR response procedures:
Getting 5ul respectively after the PCR reaction finishes identifies with 1% agarose gel electrophoresis.
4 construction of prokaryotic expression vector
The PCR fragment that implementation step 3 obtains is used BamHI according to the difference of restriction enzyme site, XhoI or HindIII, the XhoI double digestion, glue reclaims test kit and reclaims the purpose fragment.While corresponding double digestion prokaryotic expression carrier pET-28a (+), glue reclaim test kit recovery enzyme and cut big fragment, with the T4 ligase enzyme purpose fragment are connected with pET-28a (+) fragment, and through order-checking, enzyme is cut evaluation, obtains a plurality of positive recombinant expression vectors.
The abduction delivering of 5 protein molecular weight standards and expression are optimized
A plurality of recombinant expression vectors that implementation step 4 is obtained change over to and obtain the engineering bacteria that a tabulation reaches protein molecular weight standard in the e. coli bl21 (DE3), and be inoculated in card respectively and receive 220rpm/min in the LB liquid nutrient medium of penicillium mould, 37 ℃ of overnight cultures, added sec.-propyl-β-D-galactoside in 1: 50 next day behind the ratio enlarged culturing 3h is 1.0mmol/L to final concentration; Respectively at inducing 3h, 5h, 7h gets behind 9h and the 11h with volume appearance; In same culture conditions, add sec.-propyl-β-D-galactoside and divide 0.2,0.5 simultaneously to final concentration; 0.8 1.1mmol/L gets after inducing 5h with volume appearance simultaneously; 12000rmp/min, centrifugal 4min, deposition thalline.Resuspended with the 1*SDS-PAGE sample-loading buffer, boiling water boils 5-10min centrifuging and taking supernatant in the analysis of albumen polyacrylamide gel electrophoresis, thereby confirms the righttest expression time and optimum inductor final concentration.
6. the evaluation of protein expression form
According to optimum induction time that obtains in the implementation step 5 and the righttest inductor final concentration; Carry out the optimum of engineering bacteria and induce the back to collect bacterial sediment with phosphoric acid buffer PBS (pH8.0), 1%TritonX-100 is resuspended, ice bath; Carrying out ultrasonic bacteria breaking 10min; The high speed frozen centrifugation is also collected respectively and is gone up cleer and peaceful deposition, carries out the analysis of albumen polyacrylamide gel electrophoresis, identifies its expression-form.
The separation and purification of 7 target proteins
The albumen supernatant that is accredited as soluble form according to implementation step 6 combines the nickel post, washs foreign protein with phosphoric acid buffer PBS (pH8.0) earlier, uses to contain concentration as the imidazoles phosphoric acid buffer wash-out of 150mmol/L and collect target protein again; Its deposition of albumen that is accredited as the inclusion body form is followed with phosphoric acid buffer PBS (PH8.0) earlier with NaCl solution (100mmol) washed twice,
The resuspended carrying out ultrasonic bacteria breaking 2min of 1%Triton X-100 uses inclusion body washings washed twice again, uses the resuspended carrying out ultrasonic bacteria breaking 2min of inclusion body washings then, at last with inclusion body washings washing three times; The target protein that obtains is protein molecular weight standard, and each proteic molecular weight is respectively 15KD; 30KD, 45KD, 60KD; 75KD, 90KD, 120KD.
The detection of 8 target protein his labels
The his labelled detection reagent box that protein behind the purifying is used pierce company to be provided detects, and step is following: after purified proteins is carried out the SDS-polyacrylamide gel electrophoresis, and gel fixing 1h in destainer, ultrapure washing 2 times; 100ml/ time, 20min/ time, add 6xHis Tag Stain staining fluid 50ml dyeing 6min; Add ultrapure washing twice, 100ml/ time, 20min/ time; Add 50ml 6xHis Tag Developer solution, gentle concussion 15min, twice of ultrapure washing; 100ml/ time, 20min/ time, the gel imaging system ultraviolet is taken pictures.。
Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (1)
1. method of utilizing prokaryotic expression system to prepare protein molecular weight standard is characterized in that may further comprise the steps:
(1) selecting lower molecular weight standard commonly used is initiation protein molecular weight standard A, and middle molecular weight of albumen is pressed A+15, and A+30, A+45, A+60, A+75, terminal point albumen press the A+105 molecular weight standard and set; The protein molecular weight standard of setting increases progressively by 15 or 30 kilodalton gradients from initial albumen, and evenly, the gradient that wherein increases progressively between lower molecular weight is 5-15kD at interval, and the gradient that increases progressively between HMW is 30kD;
(2) selecting the low gene order of perhaps not having rare codon of rare codon content is template; Sequence signature through Expasy tools software analysis truncated gene coded protein; And the design primer obtains the different purpose fragment successively; The molecular weight that makes aim sequence be connected the coding region, back with the pET28a carrier is to set molecular weight in (1), and the righttest iso-electric point is between 4-9; Utilize the ATG of carrier simultaneously, final expressed proteins N end has the his label;
(3) after the different purpose gene that step (2) is obtained inserts expression vector pET-28a (+) according to certain restriction enzyme site, change e. coli bl21 (DE3)-star over to, obtain the engineering bacteria of a series of marking protein molecular weight standards;
The engineering bacteria of expressing a plurality of molecular weight respectively that (4) will build carries out the optimization of induction time and inductor final concentration respectively; The engineering bacteria of described a series of marking protein molecular weight standards is inoculated in respectively contains 50 μ g/ml cards and receive in the LB liquid nutrient medium of mycin 220rpm, 37 ℃ of overnight cultures; Ratio enlarged culturing 3h after added sec.-propyl-β-D-galactoside in 1: 50 next day is 1.0mmol/L to final concentration, respectively at inducing 3h, 5h; 7h; Get with volume appearance, 12000rmp centrifugal 4min, deposition thalline behind 9h and the 11h; Resuspended with the 1*SDS-PAGE sample-loading buffer, boiling water boils 5-10min centrifuging and taking supernatant and analyzes in SDS-PAGE, confirms optimum induction time; The engineering bacteria of a series of marking protein molecular weight standards is inoculated in 220rpm in the LB liquid nutrient medium that contains 50 μ g/ml kantlex respectively, and 37 ℃ of overnight cultures added sec.-propyl-β-D-galactoside in 1: 50 and be respectively 0.2 to final concentration next day behind the ratio enlarged culturing 3h; 0.5; 0.8 1.1mmol/L gets same volume, 12000rmp simultaneously after inducing 5h; Centrifugal 4min, the deposition thalline; Resuspended with the 1*SDS-PAGE sample-loading buffer, boiling water boils 5-10min centrifuging and taking supernatant and analyzes in SDS-PAGE, confirms the righttest inductor final concentration;
(5) different albumen are carried out the evaluation of expression-form; With described optimum induction time and the righttest inductor final concentration; The optimum that is applied to engineering bacteria is induced, and it is resuspended with phosphoric acid buffer and 1%TritonX-100 to collect bacterial sediment, ice bath; Carrying out ultrasonic bacteria breaking 10min; The high speed frozen centrifugation is also collected respectively and is gone up cleer and peaceful deposition, carries out SDS-PAGE in a small amount and analyzes, and identifies proteic expression-form;
(6) according to the difference of the expression-form of target protein respectively with affinitive layer purification and inclusion body purification method purifying target protein, be protein molecular weight standard; The said albumen supernatant that is accredited as soluble form that obtains is combined cobalt post Talon resin, wash foreign protein with PBS earlier, again with containing the PBS wash-out of 150mmol/L imidazoles and collecting target protein; Its deposition of albumen that is accredited as the inclusion body form is earlier with twice of NaCl solution washing; Then with phosphoric acid buffer PBS and the resuspended carrying out ultrasonic bacteria breaking 2min of 1%TritonX-100; Use inclusion body washings washed twice again, use inclusion body washings carrying out ultrasonic bacteria breaking 2min then, at last with inclusion body washings washing three times; Obtain target protein, be protein molecular weight standard;
The recombinant protein label that prokaryotic expression system is expressed comprises the antibodies district of His-tag, GST tag, MBP tag, Strep tag and staphylococcus aureus protein A; The protein molecular weight standard that correspondence produces is hybridized the testing process demonstration at western simultaneously with the albumen to be detected with label of the same race; Whether both indicated proteic molecular weight to be detected and content, it is correct also to detect the hybridization program simultaneously.
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| CN103336124B (en) * | 2012-12-11 | 2015-07-15 | 武汉工业学院 | Method and kit for detecting prion protein (PrP<SC>) |
| CN103834677B (en) * | 2014-03-26 | 2017-03-22 | 黄文林 | Recombinant expression method for human cryptochrome protein I (hCRY1) and application of human cryptochrome protein I (hCRY1) in preparation of radiotherapy protective agent |
| CN104818290A (en) * | 2015-01-28 | 2015-08-05 | 华侨大学 | Protein molecular weight standard preparation method |
| CN104862332A (en) * | 2015-06-05 | 2015-08-26 | 武汉华美生物工程有限公司 | Preparation method of protein exposure marker |
| CN105968182A (en) * | 2016-06-03 | 2016-09-28 | 黄文林 | Production process of recombinant human cryptochrome protein I (hCRY1) and composition thereof |
| CN111172185A (en) * | 2020-02-19 | 2020-05-19 | 西北农林科技大学 | Prokaryotic expression vector and construction method and application thereof |
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| US20040253620A1 (en) * | 2003-04-30 | 2004-12-16 | Invitrogen Corporation | Use of site specific recombination to prepare molecular markers |
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| CN101021538A (en) * | 2007-03-16 | 2007-08-22 | 天津科技大学 | Technique utilizing wheat glutenin as protein molecular weight standard |
| CN101281201A (en) * | 2008-05-07 | 2008-10-08 | 南京大学 | Method using implicit peptides trans splicing to manufacture protein molecular weight standard |
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