CN101531708A

CN101531708A - Soil protein extraction and intracellular protein separation method

Info

Publication number: CN101531708A
Application number: CN200910111462A
Authority: CN
Inventors: 林文雄; 熊君; 林辉锋
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2009-04-14
Filing date: 2009-04-14
Publication date: 2009-09-16
Anticipated expiration: 2029-04-14
Also published as: CN101531708B

Abstract

The invention provides a high-efficiency soil protein extraction method, comprising the following steps: adding soil in a pre-cooling soil protein extraction buffer solution and blending and oscillating the mixture in a 4 DEG C shaking table over night; performing drawing and filtering of the shaken mixture in a buchner funnel using a crude filter paper; processing the drawing and filtering liquid by macroporous resin (D101), filtering the processed liquid through a 0.22 Mum microporous membrane, the filtered liquid being the extracellular protein mother liquid; processing the cells on the filter membrane by cleaning, crushing, centrifugalizing and vacuum drying to obtain the dried powder, namely intracellular soil protein sediment. The extracellular protein mother liquid is processed by centrifuging, separating and vacuum drying to obtain the intracellular soil protein dried powder; degrading the intracellular soil protein dried powder until the degraded intracellular soil protein dried powder is clearly separated on 10% polyacrylamide gel SDS-PAG electrophoresis, the method has high separation efficiency, microbial yield is 37.5%, which is higher than the yield of the traditional method 0.1-1%, simple operation flow, good SDS-PAGE resolution and good mass spectrum identification effect.

Description

The separation method of soil proteins extraction and intracellular protein

Technical field

The present invention relates to a kind of biological technical field soil proteins extraction and the isolating method of intracellular protein.

Technical background

Grand proteomics is an emerging in recent years conception of species and investigative technique, but the microbial population that this technology is removed in the environment is studied, also can analyse in depth: can form microflora and its functional cohesion by grand proteome research, thereby better understand microflora microflora.Though the research of the grand proteomics of now having carried out seldom, but research fields such as functional gene expression, the proteinic exploitation of specific function, ecological element circulation in special extreme environment, grand proteomics has tentatively been showed its strong functions.

People such as Wilmes are research object with the active sludge with biological phosphorus removal functional first, study the variation of active sludge protein group in the complete waste water dephosphorization treating processes, represented the huge effect of grand proteomic techniques aspect the specific function of exploring environmental microorganism.People such as Schulze have studied and have been located away from dissolved organic matter in forest lake and the soil (Dissolved organic matter, range protein DOM) wish to find the extracellular protein relevant with ecological carbon cycle.Their work is first grand proteomics to be applied on the circulating research of ecological element, has obtained a lot of interesting results, has shown that grand proteomics research is in the effect that discloses on the ecological environment function.The people such as Banfield in California, USA university Berli gram branch school successfully combine the research of grand proteomics and grand genomics the extreme environment microorganism are studied, successfully utilize its achievement in research to make up the metabolic cycles mode chart of energy and material under this extreme environment, fully shown grand genomics and grand proteomics great power in conjunction with research.Yet have the general environmental sample protein extracting method that is applied to now or not, particularly from complex environment samples such as seawater, soil, extract and also have very big difficulty.Existing research such as AMD microbial film and active sludge, these samples come from extreme environment, microbial diversity is lower, for seawater, this class circumstance complication such as soil, the sample that microbial diversity is high, traditional flat band method can only provide the microorganism information less than 1%, and it is existing by widely used 1993 (O.A.Ogunseitan of grand proteome research person, 1993) method of Jian Liing, we are through repeating, do not obtain the ideal effect, show that this method lacks good versatility, therefore development is efficient, general protein extracting method is most important with application to grand proteomics research.

Summary of the invention

The purpose of this invention is to provide a kind of protein extracting method of soil efficiently, can bring up to 37.5% the protein component yield.

A kind of protein extracting method of soil efficiently comprises sample pre-treatments, proteic extraction, and concrete grammar is as follows:

Sample pre-treatments: take by weighing sample soil 65g in Erlenmeyer flask, add the soil proteins extraction damping fluid 150ml of precooling, rearmounted 4 ℃ of mixing, shaking table shaken overnight.Vibration liquid, is got suction filtration liquid and is crossed macroporous resin (D101) in the B vacuum filtration with filter paper, gets post liquid and crosses 0.22 μ m millipore filtration, and filtrate is the exoprotein mother liquor, does further intracellular protein on the filter membrane and extracts.

Soil proteins extraction damping fluid proportioning: 0.82g potassium primary phosphate+0.16g dipotassium hydrogen phosphate is settled to 100ml, to wherein adding 8ml0.5M, pH8.0EDTA.

The extraction of exoprotein: get the exoprotein mother liquor, the acetone soln that adds isopyknic precooling staticly settles 4-5h, 30000rpm behind the mixing, 4 ℃ of centrifugal 30min, abandon supernatant, the precipitation washing with acetone of precooling, 13000rpm, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 10min, 13000rpm with the dehydrated alcohol of precooling under 4 ℃ of ultrasonic wave, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 13000rpm with the ether of precooling, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation uses Vacuumdrier drying, dry powder to be the outer soil proteins precipitate of born of the same parents.

The extraction of intracellular protein: get above-mentioned filtering filter membrane in beaker, add the soil proteins extraction damping fluid of the precooling of 20ml, in 4 ℃ of supersound washings of ultrasonic instrument 10 minutes, abandon filter membrane, solution is poured in the 50ml centrifuge tube, centrifuge tube cytoclasis in the cytoclasis instrument (power 15%, time 600s, 4 ℃ of groove temperature, ultrasonic interval 5s), after cytoclasis is finished, in centrifuge tube, add the acetone soln of isopyknic precooling, staticly settle 4-5h behind the mixing, 30000rpm, 4 ℃ of centrifugal 30min abandon supernatant, the precipitation washing with acetone of precooling, 13000rpm, 4 ℃ of centrifugal 10min abandon supernatant, precipitation is washed 10min with the dehydrated alcohol of precooling under 4 ℃ of ultrasonic wave, 13000rpm, 4 ℃ of centrifugal 10min abandon supernatant, precipitation is washed with the ether of precooling, 13000rpm, 4 ℃ of centrifugal 10min abandon supernatant, precipitation uses Vacuumdrier drying, dry powder to be soil proteins precipitate in the born of the same parents.

The separation method of intracellular protein:

Sample dissociation: take by weighing intracellular protein dry powder ratio CHAPS extracting solution 20 ℃ of supersound process 10 * 1.5min in ultrasonic cleaner in 15 μ l/mg, each supersound process interim concussion 30S, 25 ℃ of centrifugal 20min of 12000rpm, it is standby to get supernatant.CHAPS extracting solution proportioning: 9M urea, 1% dithiothreitol (DTT) (DTT), 4%CHAPS, 2%Ampholine (pH5-8).

The SDS-PAGE electrophoresis: the gel specification is 102 * 84 * 2.4mm, and separation gel is: 10% polyacrylamide gel, seal with 1% agarose (electrophoretic buffer configuration); Electrophoresis chamber adds electrode buffer; The 100V constant voltage is carried out electrophoresis, and room temperature treats that tetrabromophenol sulfonphthalein can stop electrophoresis from bottom 1cm, electrophoresis 2.5 hours; Electrophoresis chamber electrode buffer proportioning: 25mmol/L Tris, 192mmol/L glycine, 0.1%SDS.

Experimental results show that repeatedly 10% polyacrylamide gel is better to the proteic resolving effect of soil, reach and to know the degree of differentiating.

The amount that goes up earth proteins extraction damping fluid and classes of agents in the aforesaid method can be amplified in proportion according to the amount of soil, but proportioning, method and processing condition are constant.

Adopt method provided by the present invention to have outstanding characteristics:

1, adopt the macropore resin to filter the organic matter that can hold back in the pedotheque, and microorganism and extracellular protein can not with the covalent bonds in the resin, thereby can not be adsorbed by the macropore resin, and by wash-out in damping fluid.This method can not only improve the extraction yield of soil microorganisms, also can improve electrophoresis, the separation efficiency of protein electrophorese simultaneously.

2, the soil protein yield of being extracted is about 37.5%. to similar by the yield of acridine orange staining cell counting gained microorganism.

3,10% polyacrylamide gel is better to the proteic resolving effect of soil, reaches to know the degree of differentiating.

Specific embodiment:

Take by weighing sample soil 65g in Erlenmeyer flask, add the soil proteins extraction damping fluid 150ml of precooling, rearmounted 4 ℃ of mixing, shaking table shaken overnight.Vibration liquid, is got suction filtration liquid and is crossed macroporous resin (D101) in the B vacuum filtration with filter paper, gets post liquid and crosses 0.22 μ m millipore filtration, and filtrate is the exoprotein mother liquor, does further intracellular protein on the filter membrane and extracts.

The preparation of soil proteins extraction damping fluid: 0.82g potassium primary phosphate+0.16g dipotassium hydrogen phosphate is settled to 100ml, to wherein adding 8ml0.5M, pH8.0EDTA.

The outer soil protein dry powder of born of the same parents is put-20 ℃ of refrigerator preservations a middle or short term, is preserved in for a long time in-80 ℃ of refrigerators.

The extraction of intracellular protein: get above-mentioned filtering filter membrane in beaker, the soil proteins extraction damping fluid that adds the precooling of 20ml, in 4 ℃ of supersound washings of ultrasonic instrument 10 minutes, abandon filter membrane, solution is poured in the 50ml centrifuge tube, centrifuge tube cytoclasis in the cytoclasis instrument, cytoclastic condition: 1000s, interval 5s, power output 15%, 4 ℃ of temperature.After cytoclasis is finished, in centrifuge tube, add the acetone soln of isopyknic precooling, staticly settle 4-5h behind the mixing, 30000rpm, 4 ℃ of centrifugal 30min, abandon supernatant, the precipitation washing with acetone of precooling, 13000rpm, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 10min, 13000rpm with the dehydrated alcohol of precooling under 4 ℃ of ultrasonic wave, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 13000rpm with the ether of precooling, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation uses Vacuumdrier drying, dry powder to be soil proteins precipitate in the born of the same parents.

The soil protein dry powder is put-20 ℃ of refrigerator preservations a middle or short term in the born of the same parents, is preserved in for a long time in-80 ℃ of refrigerators.

The separation of intracellular protein:

Intracellular protein cracking: take by weighing above-mentioned intracellular protein dry powder and add the CHAPS extracting solution in the ratio of 15 μ l/mg, 20 ℃ of supersound process 10 * 1.5min in ultrasonic cleaner, each supersound process interim is shaken 30 S, 25 ℃ of centrifugal 20min of 12000rpm, and it is standby to get supernatant.

CHAPS extracting solution proportioning: 9M urea, 1% dithiothreitol (DTT) (DTT), 4%CHAPS, 2%Ampholine (pH5-8).

The SDS-PAGE electrophoresis: the gel specification is 102 * 84 * 2.4mm, and separation gel is: 10% polyacrylamide gel, seal with 1% agarose; Electrophoresis chamber adds electrode buffer, and the 100V constant voltage is carried out electrophoresis, and room temperature treats that tetrabromophenol sulfonphthalein can stop electrophoresis from bottom 1cm, electrophoresis 2.5 hours.

The electrode buffer proportioning of electrophoresis chamber: 25mmol/L Tris, 192mmol/L glycine, 0.1%SDS.

Then sample is delivered to Shanghai proteomics test center and carried out the LC-MS-MS analysis with identification of protein kind and source.

Claims

1, a kind of protein extracting method of soil efficiently comprises sample pre-treatments, proteic extraction, it is characterized in that:

Sample pre-treatments: take by weighing sample soil 65g in Erlenmeyer flask, add the soil proteins extraction damping fluid 150ml of precooling, rearmounted 4 ℃ of mixing, shaking table shaken overnight; Vibration liquid, is got suction filtration liquid and is crossed macroporous resin (D101) in the B vacuum filtration with filter paper, gets post liquid and crosses 0.22 μ m millipore filtration, and filtrate is the exoprotein mother liquor, does further intracellular protein on the filter membrane and extracts;

The preparation of soil proteins extraction damping fluid: 0.82g potassium primary phosphate+0.16g dipotassium hydrogen phosphate is settled to 100ml, to wherein adding 8ml 0.5M, pH8.0 EDTA;

The extraction of exoprotein: get the exoprotein mother liquor, the acetone soln that adds isopyknic precooling staticly settles 4-5h, 30000rpm behind the mixing, 4 ℃ of centrifugal 30min, abandon supernatant, the precipitation washing with acetone of precooling, 13000rpm, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 10min, 13000rpm with the dehydrated alcohol of precooling under 4 ℃ of ultrasonic wave, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 13000rpm with the ether of precooling, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation uses Vacuumdrier drying, dry powder to be the outer soil protein of born of the same parents;

The extraction of intracellular protein: get above-mentioned 0.22 μ m millipore filtration in beaker, add the last earth proteins extraction damping fluid of the precooling of 20ml, in 4 ℃ of supersound washings of ultrasonic instrument 10 minutes, abandon filter membrane, solution is poured in the 50ml centrifuge tube, centrifuge tube cytoclasis in the cytoclasis instrument, after cytoclasis is finished, in centrifuge tube, add the acetone soln of isopyknic precooling, staticly settle 4-5h behind the mixing, 30000rpm, 4 ℃ of centrifugal 30min, abandon supernatant, the precipitation washing with acetone of precooling, 13000rpm, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 10min, 13000rpm with the dehydrated alcohol of precooling under 4 ℃ of ultrasonic wave, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is washed 13000rpm with the ether of precooling, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation uses Vacuumdrier drying, dry powder to be soil proteins precipitate in the born of the same parents.

2, a kind of separation method of the intracellular protein of soil efficiently comprises cracking of soil intracellular protein and SDS-PAGE electrophoresis, it is characterized in that:

Intracellular protein cracking: take by weighing intracellular protein dry powder that claim 1 method obtains and add the CHAPS extracting solution, 20 ℃ of supersound process 10 * 1.5min in ultrasonic cleaner, each supersound process interim concussion 30S in the ratio of 15 μ l/mg; 25 ℃ of centrifugal 20min of 12000rpm, it is standby to get supernatant; CHAPS extracting solution proportioning is: 9M urea, 1% dithiothreitol (DTT) (DTT), 4%CHAPS, 2%Ampholine (pH5-8);

The SDS-PAGE electrophoresis: the gel specification is 102 * 84 * 2.4mm, and separation gel is: the 9-12% polyacrylamide gel, seal with 1% agarose (electrophoretic buffer configuration); Electrophoresis chamber adds electrode buffer: 25mmol/L Tris, 192mmol/L glycine, 0.1%SDS; The 100V constant voltage is carried out electrophoresis, and room temperature treats that tetrabromophenol sulfonphthalein can stop electrophoresis from bottom 1cm, electrophoresis 2.5 hours.

3, the separation method of a kind of intracellular protein of soil efficiently according to claim 2, when it is characterized in that the SDS-PAGE electrophoresis, adopting 10% polyacrylamide gel is separation gel.