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Mammalian cell tandem affinity purification vector and method for purifying protein complex thereof

A carrier and affinity technology, applied in the field of protein separation and purification, to reduce the influence of function or stability, and the effect of reducing the molecular weight of tags

Inactive Publication Date: 2005-09-28
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the pBS1761, pBS1479 and other plasmid vectors containing the TAP tag (TAP tag) originally constructed by Rigaut are suitable for isolating protein complexes from yeast, but for higher eukaryotic cells and mammalian cells, it is difficult to directly use these TAP tag vectors to effectively purify the target protein complex

Method used

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  • Mammalian cell tandem affinity purification vector and method for purifying protein complex thereof

Examples

Experimental program
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Embodiment 1

[0029] Example 1 Construction of tandem affinity purification vector p2FCBP

[0030] The tandem affinity purification vector is developed on the basis of the retrovirus vector MSCVpac. The TAP tag sequence composed of 2×Flag, TEV enzyme digestion sequence and CBP sequence plus the linker sequence of HindIII and BamH I restriction endonucleases, and the vector that was double-digested with restriction endonucleases HindIII and BamH I MSCVpac is connected.

[0031] Prepare tandem affinity purification vectors such as figure 1 As shown, named p2FCBP. The p2FCBP vector has multiple cloning sites such as BamH I, BstX I, EcoRV, and Xho I for convenient insertion of foreign genes, and two identical 8-peptide Flag tags (DFlag) and one Calmodulin-binding peptide (CBP) sequence, there is a TEV enzyme cleavage site between the Flag tag and the CBP sequence. There is also a screening marker encoding ampicillin (Amp) and puromycin N-acetyl transferase (puromycin N-acetyl transferase) o...

Embodiment 2

[0032] Example 2 Application of the p2FCBP vector to identify the interacting protein of the target protein 14-3-3ε in QGY7703 cells

[0033] (1) Cell culture and collection

[0034] QGY7703 was cultured in culture medium 1640 containing 10% fetal calf serum (FCS) at 37°C and 5% CO 2 , passaging every 2-3 days. When the cells grow to 80-100%, treat with 0.25% trypsin / 0.02% EDTA digestion solution for a few minutes to detach the cells, add pre-cooled PBS to suspend, centrifuge to wash off the culture medium, and repeat with PBS again.

[0035] (2) RT-PCR

[0036] 10 will be collected 6 Extract total RNA from QGY7703 cells and use 50 μl RNase-free ddH 2 O dissolved. The sense primer (5'-TATGGATCCGATGATCGAGAGGATCTGGTGTAC-3') for PCR amplification of 14-3-3ε; the antisense primer (5'-ATTCTCGAGTCACTGATTTTCGTCTTCCACGTC-3'). RT-PCR conditions are: 2 μl of total RNA as template, 1 μl of 20 μmol / L primers, 5 μl of 10 mmol / L dNTP, 25 mmol / L MgCl 2 10μl, 5μl of 10×PCR buffer, 1μl ...

Embodiment 3

[0047] Example 3 Application of the p2FCBP vector to identify the interacting protein of the target protein 14-3-3ε in MHCC97H cells

[0048] The steps are the same as in Example 2, except that the p2FCBP recombinant vector inserted with 14-3-3ε cDNA is transferred into MHCC97H cells for fusion protein expression. Final protein profiling revealed that the components in the 14-3-3ε complex were 14-3-3β and keratin CK 1.

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Abstract

The present invention belongs to the field of protein separating and purifying technology, and provides one kind of tandem affinity purified vector for mammal cell and its preparation process and usage in purifying protein composition. Tandem affinity purification (TAP) is used in separating protein composition and researching protein-protein interaction in physiological condition. The present invention has two Flag sequences to replace the protein A flag sequence in initial TAP vector and the decreased flag molecular weight is favorable to the effective fusion expression between the foreign target gene and the TAP vector and the high affinity combination to the Flag resisting monoclonal antibody. Therefore, the TAP vector is used in separating protein composition of higher mammal cell effectively and simply.

Description

technical field [0001] The invention belongs to the field of protein separation and purification. Specifically, the invention provides a mammalian cell (mammalian cell) tandem affinity purification carrier, a preparation method thereof and a method for purifying a protein complex. The application of the tandem affinity purification carrier system can efficiently separate protein complexes and study the physical interaction between proteins. Background technique [0002] With the completion of the whole genome sequencing of many organisms, life science has entered the post-genome era of major research contents such as genome and proteome, and a new discipline of proteomics has also emerged as the times require. The analysis of protein-protein interaction is a new hot topic in proteomics, analytical chemistry, biochemistry and other interdisciplinary research. The huge amount of data generated by large-scale genome sequencing now urgently requires systems proteomics to elucid...

Claims

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Application Information

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IPC IPC(8): C07K1/14C12N15/65C12N15/85C12P21/02
Inventor 梁淑芳王天翼陈先杨芃原陆豪杰
Owner FUDAN UNIV
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