CN103275945A - Method for preparing soluble HIV-1 integrase recombinant protein - Google Patents
Method for preparing soluble HIV-1 integrase recombinant protein Download PDFInfo
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- CN103275945A CN103275945A CN2013101882885A CN201310188288A CN103275945A CN 103275945 A CN103275945 A CN 103275945A CN 2013101882885 A CN2013101882885 A CN 2013101882885A CN 201310188288 A CN201310188288 A CN 201310188288A CN 103275945 A CN103275945 A CN 103275945A
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Abstract
The invention discloses a method for preparing a soluble HIV-1 integrase recombinant protein, and belongs to the technical field of biology. The preparation method comprises the steps of induced expression, bacteria disruption, purification and the like. The adopted expression medium contains 15 to 35 g/L of tryptone, 10 to 20 g/L of yeast extract, 2 to 3 g/L of sodium chloride, 1 g/L of glucose, 10 to 15 g/L of glycerin, 2.05 g/L of disodium hydrogen phosphate, 1.27 g/L of sodium dihydrogen phosphate, 50 mg/L of magnesium sulfate, and 60 mg/L of kanamycin; and the adopted cracking buffer solution contains 20 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 1 M of sodium chloride, 2 mM of beta-mercaptoethanol, 0.3 mg/ml of lysozyme and 5 mM of imidazole, and the pH value of the buffer solution is 9.0. The expression medium can ensure normal growth of host bacteria and efficient expression of the objective protein; and the cracking buffer solution can enhance the disruption effect of bacteria, improve the solubility of the objective protein and finally improve the yield of the objective protein.
Description
Technical field
The invention belongs to biological technical field, specifically relate to a kind of preparation method of solubility HIV-1 intergrase recombinant protein.
Background technology
The HIV-1 intergrase comprises 288 amino-acid residues by HIV-1 pol genes encoding, and molecular weight is about 32 kDa.Intergrase catalysis is incorporated in the genome of host cell through the HIV-1 cDNA that reverse transcription forms, and is the essential link in the HIV-1 replicative cycle.Because do not have the functional analogue of intergrase in the human body cell, the inhibitor that acts on intergrase is very little to the potential side effect of human body, so intergrase is considered to the desirable target spot of anti-HIV-1 drug research.
In HIV-1 intergrase relevant mechanism and drug research, need prepare the intergrase recombinant protein with the means of prokaryotic expression purifying usually.Yet, when preparing the intergrase recombinant protein with existing prokaryotic expression means of purification, ubiquity target protein poorly soluble, easily form inclusion body, problem that productive rate is low, follow-up study is caused to a certain degree influence.
Summary of the invention
The purpose of this invention is to provide that a kind of step is simple, rapid and convenient and HIV-1 intergrase chain transfer reaction inhibitor in-vitro screening method with low cost.
The preparation method of a kind of solubility HIV-1 intergrase recombinant protein provided by the present invention may further comprise the steps:
(1) will be with pET-28a(+) carrier is that the intergrase expression of recombinant proteins carrier of fundamental construction is transformed into e. coli bl21 (DE3) competent cell, the picking positive colony is inoculated in the LB liquid nutrient medium that 5 ml contain 50 mg/L kantlex, and 37 ℃ of shaking table shaking culture 12-16 h obtain culture;
(2) with the culture in (1) by volume 1 % be inoculated in and express in the substratum, 37 ℃ of shaking table shaking culture to bacterium liquid OD600 values reach 0.8, the adding isopropyl-is 0.4 mM to the final concentration of isopropyl-, and 37 ℃ of shaking table shaking culture 4-5 h obtain culture; Described expression substratum contains 15-35 g/L Tryptones, 10-20 g/L yeast extract, 2-3 g/L sodium-chlor, 1 g/L glucose, 10-15 g/L glycerine, 2.05 g/L Sodium phosphate dibasics, 1.27 the g/L SODIUM PHOSPHATE, MONOBASIC, 50 mg/L sal epsom, 60 mg/L kantlex;
(3) with the centrifugal precipitation that obtains of the culture in (2), per 100 mg precipitation is resuspended with 1 ml lysis buffer, incubated at room 30 min, and multigelation places ice bath ultrasonication 10 min, 4 ℃ of centrifugal collection supernatant liquors afterwards twice; Described lysis buffer contains 20 mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1 M sodium-chlor, and 2 mM beta-mercaptoethanols, 0.3 mg/ml N,O-Diacetylmuramidase, 5 mM imidazoles are with sodium hydroxide adjust pH to 9.0;
(4) supernatant liquor in (3) is carried out nickel sepharose affinity chromatography, carry out gradient elution with the washing lotion that contains 20 mM, 50 mM, 100 mM, 250 mM imidazoles successively, with the elutriant dialysis desalination of collecting that contains target protein, and carry out packing.
As a kind of preferred version, the preparation method of described a kind of solubility HIV-1 intergrase recombinant protein, expression substratum in the step (2) contains 25 g/L Tryptoness, 15 g/L yeast extracts, 2.5 g/L sodium-chlor, 1 g/L glucose, 12 g/L glycerine, 2.05 g/L Sodium phosphate dibasics, 1.27 g/L SODIUM PHOSPHATE, MONOBASIC, 50 mg/L sal epsom, 60 mg/L kantlex.
Express each composition of substratum, lysis buffer among the present invention and all can buy from biochemical reagents company, and can be formulated according to the ordinary method that those skilled in the art understand thoroughly.
The inventive method has following beneficial effect:
Traditional substratum is transformed, through test in a large number, obtained the expression substratum of optimizing.Each composition of expressing in the substratum can guarantee the normal growth of host bacterium and efficiently expressing of target protein, and can increase solubility and the stability of target protein.Wherein, the glucose of 1 g/L can stop the too early expression of target protein; The glycerine of 10-15 g/L can be used as the good sources of carbon in abduction delivering later stage, promotes the expression of target protein; 2.05 can making substratum maintain, the SODIUM PHOSPHATE, MONOBASIC of the Sodium phosphate dibasic of g/L and 1.27 g/L is beneficial to the pH environment that thalli growth and target protein are expressed.The higher pH value of lysis buffer can be strengthened the crushing effect of thalline, increases the solubility of target protein, finally improves the productive rate of target protein.Use the inventive method, need not the complicated procedures such as sex change renaturation through inclusion body, can directly supernatant liquor be carried out affinity chromatography behind the bacterial cell disruption, obtain great amount of soluble HIV-1 intergrase recombinant protein.In addition, induce mode when the inventive method also need not traditional low temperature length, shortened the preparation time of target protein, improved working efficiency.
Description of drawings
Fig. 1 is 15% SDS-polyacrylamide gel electrophoresis experimental result, three row are followed successively by supernatant liquor behind the existing method bacterial cell disruption, the supernatant liquor behind the inventive method bacterial cell disruption, the band of target protein from left to right, and the band that is gone out by the square frame frame is respectively the target protein band in the supernatant liquor behind existing method, the inventive method bacterial cell disruption.
Embodiment
The invention will be further described below in conjunction with embodiment:
Prepare solubility HIV-1 intergrase recombinant protein according to following steps:
(1) will be with pET-28a(+) carrier is that the intergrase expression of recombinant proteins carrier pWIN of fundamental construction is transformed into e. coli bl21 (DE3) competent cell, the picking positive colony is inoculated in the LB liquid nutrient medium that 5 ml contain 50 mg/L kantlex, and 37 ℃ of shaking table shaking culture 14 h obtain culture.
(2) with the culture in (1) by volume 1 % be inoculated in 0.6 L and express in the substratum, 37 ℃ of shaking table shaking culture to bacterium liquid OD600 values reach 0.8, the adding isopropyl-is 0.4 mM to the final concentration of isopropyl-, and 37 ℃ of shaking table shaking culture 4.5 h obtain culture; Express and contain 25 g/L Tryptoness (available from the silent generation that company that flies of U.S.'s match) in the substratum, 15 g/L yeast extracts (available from the silent generation that company that flies of U.S.'s match), 2.5 g/L sodium-chlor, 1 g/L glucose, 12 g/L glycerine, 2.05 g/L Sodium phosphate dibasics, 1.27 g/L SODIUM PHOSPHATE, MONOBASIC, 50 mg/L sal epsom, 60 mg/L kantlex.
(3) with centrifugal bacterial sediment 2.0 g that obtain of the culture in (2), per 100 mg precipitation is resuspended with 1 ml lysis buffer, incubated at room 30 min, and multigelation places ice bath ultrasonication 10 min, 4 ℃ of centrifugal collection supernatant liquors afterwards twice; Contain 20 mM 4-hydroxyethyl piperazine ethanesulfonic acid in the lysis buffer, 1 M sodium-chlor, 2 mM beta-mercaptoethanols, 0.3 mg/ml N,O-Diacetylmuramidase (available from Merck KGaA company), 5 mM imidazoles are with sodium hydroxide adjust pH to 9.0.15% SDS-polyacrylamide gel electrophoresis experimental result shows that the content of target protein significantly improves (see figure 1) in the supernatant liquor behind the inventive method bacterial cell disruption.As calculated, compare with existing solubility intergrase preparation method, total protein content improves 28% in the supernatant liquor behind the inventive method bacterial cell disruption, and the content of target protein improves 67%, show that the inventive method strengthened the crushing effect of thalline, increased the solubility of target protein.
(4) supernatant liquor in (3) is carried out nickel sepharose affinity chromatography, carry out gradient elution with the washing lotion that contains 20 mM, 50 mM, 100 mM, 250 mM imidazoles successively, with the elutriant dialysis desalination of collecting that contains target protein, and carry out packing.Obtain 15 mg solubility integrase proteins altogether, and adopt existing solubility intergrase preparation method, at most only can obtain 9 mg solubility integrase proteins, the productive rate of the inventive method target protein has improved nearly one times.
Claims (2)
1. the preparation method of a solubility HIV-1 intergrase recombinant protein is characterized in that may further comprise the steps:
(1) will be with pET-28a(+) carrier is that the intergrase expression of recombinant proteins carrier of fundamental construction is transformed into e. coli bl21 (DE3) competent cell, the picking positive colony is inoculated in the LB liquid nutrient medium that 5 ml contain 50 mg/L kantlex, and 37 ℃ of shaking table shaking culture 12-16 h obtain culture;
(2) with the culture in (1) by volume 1 % be inoculated in and express in the substratum, 37 ℃ of shaking table shaking culture to bacterium liquid OD600 values reach 0.8, the adding isopropyl-is 0.4 mM to the final concentration of isopropyl-, and 37 ℃ of shaking table shaking culture 4-5 h obtain culture; Described expression substratum contains 15-35 g/L Tryptones, 10-20 g/L yeast extract, 2-3 g/L sodium-chlor, 1 g/L glucose, 10-15 g/L glycerine, 2.05 g/L Sodium phosphate dibasics, 1.27 the g/L SODIUM PHOSPHATE, MONOBASIC, 50 mg/L sal epsom, 60 mg/L kantlex;
(3) with the centrifugal precipitation that obtains of the culture in (2), per 100 mg precipitation is resuspended with 1 ml lysis buffer, incubated at room 30 min, and multigelation places ice bath ultrasonication 10 min, 4 ℃ of centrifugal collection supernatant liquors afterwards twice; Described lysis buffer contains 20 mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1 M sodium-chlor, and 2 mM beta-mercaptoethanols, 0.3 mg/ml N,O-Diacetylmuramidase, 5 mM imidazoles are with sodium hydroxide adjust pH to 9.0;
(4) supernatant liquor in (3) is carried out nickel sepharose affinity chromatography, carry out gradient elution with the washing lotion that contains 20 mM, 50 mM, 100 mM, 250 mM imidazoles successively, with the elutriant dialysis desalination of collecting that contains target protein, and carry out packing.
2. the preparation method of a kind of solubility HIV-1 intergrase recombinant protein according to claim 1, it is characterized in that the expression substratum in the step (2) contains 25 g/L Tryptoness, 15 g/L yeast extracts, 2.5 g/L sodium-chlor, 1 g/L glucose, 12 g/L glycerine, 2.05 g/L Sodium phosphate dibasic, 1.27 the g/L SODIUM PHOSPHATE, MONOBASIC, 50 mg/L sal epsom, 60 mg/L kantlex.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003798A (en) * | 2006-01-17 | 2007-07-25 | 温州医学院 | Purified expression of recombined beta lactamase in superspectrum, and fermentation process in high density |
| CN102002517A (en) * | 2010-09-29 | 2011-04-06 | 北京正旦国际科技有限责任公司 | Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003798A (en) * | 2006-01-17 | 2007-07-25 | 温州医学院 | Purified expression of recombined beta lactamase in superspectrum, and fermentation process in high density |
| CN102002517A (en) * | 2010-09-29 | 2011-04-06 | 北京正旦国际科技有限责任公司 | Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 程绍辉等: "HIV-1整合酶蛋白的可溶性表达及功能研究", 《中国生物工程杂志》, no. 1, 31 December 2006 (2006-12-31), pages 22 - 26 * |
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