Multi-subunit protein renaturation method

A protein and subunit technology, applied in the field of biochemistry and molecular biology, can solve the problem of no chromatographic renaturation method, and achieve the effect of saving raw materials and simplifying the operation process

Inactive Publication Date: 2012-01-04
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing chromatographic renaturation technology is limited to the renaturation of single-subunit proteins with a complete peptide chain, and there is no suitable chromatographic renaturation method for proteins composed of two or more subunits, such as the major histocompatibility complex

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Amplification of MHC I heavy chain gene and construction of recombinant plasmid.

[0031]Primers were designed according to the cDNA sequence of human MHC I heavy chain in GenBank (AF036921.1GI: 4104536). At the C-terminus of the heavy chain gene, there is a BSP (biotin protein ligaseBirA substrate peptide) sequence (17aa: GSLHHILDAQKMVWNHR) for biotinylation and can be combined with Ni 2+ - NTA-bound His-tag (6aa: HHHHHH). The upstream primer contains initiation codon, Nco I recognition site and protection base, and the downstream primer contains Xho I restriction site. Their sequences are:

[0032] Upstream p1: 5`-GCCCATGGGCTCTCACTCCATGAGGTAT-3`;

[0033] Downstream p2: 5`-GCCTCGAGACGATGATTCCACACCATTTTCTGTGCATCCAGAATATGATGCAGGGATCCGGTGAGGGGCTTGGGCAA-3`.

Embodiment 2

[0036] Example 2: Extraction of human peripheral blood cell total RNA and HBc 18-27 - Construction of recombinant plasmids for β2 microglobulin.

[0037] (1) Amplification of β2 microglobulin (β2m) cDNA

[0038] The extraction steps of total RNA from human peripheral blood cells were carried out according to the method recommended by the TRIZOL kit (Promega).

[0039] Primers were designed according to the cDNA sequence of human β2m in GenBank (S71244.1GI:547297).

[0040] The upstream primer contains initiation codon, Nco I recognition site and protection base, and the downstream primer contains Xho I restriction site. Their sequences are:

[0041] Upstream p3: 5`-GGCCCATGGATATCCAGCGTACTCCAAAG-3`;

[0042] Downstream p4: 5`-CAACTCGAGattaCATGTCTCGATCCCACTTAAC-3`.

[0043] The reverse transcription "RT" system and process of the reverse transcription polymerase chain reaction "RT-PCR" used to amplify the β2 microglobulin gene are as follows: add 6 μl of DEPC water to the 2...

Embodiment 3

[0054] Example 3: MHC I heavy chain protein and HBc 18-27 -Expression and preliminary purification of β2 microglobulin

[0055] MHC I heavy chain and HBc 18-27 -β2 microglobulin recombinant plasmids were transformed into host bacteria E.coli BL21(DE3), picked clones and inoculated in LB medium containing kanamycin (35μg / ml), cultured to OD 600 0.6-0.8, add IPTG to a final concentration of 0.1mM, and induce at 37°C for 3h. Collect the bacterial pellet by centrifugation at 5000g for 10min, and resuspend in ice-cold Buffer A (20mM Tris, 150mM NaCl, pH8.0), mix well and perform high-pressure homogeneous destruction, centrifuge at 10000g at 4°C for 10min, discard the supernatant, and weigh the precipitate Suspend in Buffer B (20mM Tris, 150mM NaCl, 0.5% Triton X-100, pH8.0), mix well and centrifuge at 10000g at 4°C for 10min, discard the supernatant, and collect the inclusion body precipitate. Then repeat the wash once, resuspend the pellet in Buffer B, mix well and centrifuge. ...

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Abstract

Disclosed is a refolding method of plural- subunits protein, which is characterized in that protein is composed of two or more than two subunits; one protein subunit or plural protein subunits is or are prerefolded firstly, and the other protein subunit or other plural protein subunits is or are combined on a chromatographic column; renaturation solution containing the prerefolded protein subunits is increased by step so that the protein subunits combined on the chromatographic column can be refolded and can be combined with the prerefolded protein subunits to form a complete protein molecule; and after being washed, the refolded protein is eluted from the chromatographic column. With the refolding method to prepare the protein composed of plural subunits, the yield of the protein is high, the protein is not needed to be purified, the operation is simple, and the raw material, the time and the labor can be saved.

Description

technical field [0001] The invention belongs to the technical field of biochemistry and molecular biology, and in particular relates to a method for renaturation of multi-subunit proteins. Background technique [0002] The renaturation technology of denatured protein is an experimental technique often used in the field of modern life sciences. It means that denatured protein will spontaneously change from a denatured thermally unstable state to a thermodynamically stable state after the denaturant such as hydrochloric acid or urea is removed or the concentration is reduced. state transition, resulting in the formation of natural structures with biological functions. The commonly used refolding methods can be divided into two categories: dilution / dialysis refolding method and chromatography refolding method. Dilution / dialysis renaturation method is currently the simplest and most commonly used renaturation method, including dilution renaturation method and dialysis renaturat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22C07K1/20C07K1/18C07K1/14
Inventor 张建琼孟凡岩谢维沈传来
Owner SOUTHEAST UNIV
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