Method for preparing high-uniformity single-order streptavidin tetramer

A streptavidin and tetramer technology, applied in the field of genetic engineering, can solve the problems of reducing immune reactivity, improving uniformity, and reducing Biotin affinity, etc., achieving no recombination tags, high uniformity, and stability sexual effect

Inactive Publication Date: 2015-10-07
HUBEI UNIV OF ARTS & SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the monovalent streptavidin tetramer obtained by affinity chromatography has the advantage of having a Biotin binding site, but the disadvantage is that the uniformity needs to be improved. In addition, the exogenous purification tag used in the preparation process can reduce SA Affinity for Biotin
In the present invention, the advantages of treating monovalent streptavidin tetramers with proteases such as protease PK (proteinase K) and SU (subtilisin) are that more stable proteins can be screened out, and at the same time, exogenous purification tags (histag) can be removed To avoid its adverse effects on ligand binding, and reduce immunoreactivity, the present invention has not yet seen relevant reports

Method used

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  • Method for preparing high-uniformity single-order streptavidin tetramer
  • Method for preparing high-uniformity single-order streptavidin tetramer
  • Method for preparing high-uniformity single-order streptavidin tetramer

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Preparation of high uniformity monovalent streptavidin tetramer

[0024] (1) Streptavidin wild type (with His at the C-terminal 8 -tag tagged recombinant protein) and activity loss mutants (without His 8 -tag tag) was cloned into the NdeI and XhoI restriction sites in the pET28a vector.

[0025] (2) The recombinant plasmid was transformed into E.coli competent cells BL21(DE3), and the cells were cultured to OD 600 When =0.7, inducer IPTG was added to a final concentration of 1 mM for induction. Cells were collected after induction at 37°C for 5 hours and frozen at -20°C.

[0026] (3) Frozen cells were resuspended in a bacteriostasis buffer (20 mM Tris-HCl pH 8.0, 0.3 M NaCl) at a ratio of buffer: cell weight of 5 ml / g, and then ultrasonically disrupted in an ice bath. After the crushed homogenate was centrifuged at 4°C and 38000g for 30min, the precipitate was resuspended in washing buffer (20mM Tris-HCl pH8.0, 0.3M NaCl, 2M Urea, 2% Triton X-100), and the...

Embodiment 2

[0033] Add six different proteases (hampton research) (PE, Pepsin; PK, Proteinase K; TR, Trypsin; TH, Thermolysin; α-C, Chymotrypsin, Alpha; SU, Subtilisin) according to the mass ratio of sample / protease 100:1, Analysis was performed using SDS-PAGE (15%) after incubation for 5 hours at 37°C.

[0034] The results show that tetrameric W1M3 is resistant to all proteases tested. At the same time, the results of Western-blot analysis showed that protease PK and SU can remove exogenous His very effectively 8 -tag, which further improves the homogeneity of streptavidin tetramer W1M3 and avoids exogenous his 8 -tag-induced decrease in ligand binding ability and possible immunogenicity.

[0035]

[0036]

[0037]

[0038]

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Abstract

The invention discloses a method for preparing high-uniformity single-order streptavidin tetramer. Recombinant plasmids of wild type streptavidin and activity deletion mutants are prepared separately; engineering bacteria are further prepared; recombined streptavidin is expressed through engineering bacteria fermentation; the obtained wild type and mutant streptavidin inclusion bodies are mixed according to a mass ratio of 1:3; centrifugation is carried out; inclusion body renaturation and separation are carried out to obtain the single-order streptavidin tetramer; and an His8-tag is removed through protease K or bacillus subtilis protease. The preparing method is simple and easy to operate; the single-order streptavidin tetramer does not contain the His8-tag label which has negative effects on the activity; in addition, various factors can be borne, the state of the single-order tetramer is kept, and the method has the uniformity and the stability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing monovalent streptavidin tetramers with high uniformity and no recombination tags. Background technique [0002] Streptavidin (Streptavidin, SA) is a small molecule non-glycosylated protein secreted by Streptomyces avidinii during the culture process. The full-length coding region is 183 amino acids (aa), including 24aa Signal peptide, mature molecule 159aa. The natural active form outside the bacteria is a mature peptide from 13 to 139aa. The molecular weight of the mature molecule is 15kD. SA core sequence, with anti-protease properties. In 1963, Dr. Stapley first discovered and reported SA. Since this century, SA has been highly valued by the international biological and medical circles because of its high affinity and specificity with biotin (also known as vitamin H and coenzyme R). The affinity of SA to its ligand biotin (10 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/36
Inventor 张敏于洪军
Owner HUBEI UNIV OF ARTS & SCI
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