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Updated: Mar 28, 2026
Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
[Screening of special scFv antibody against human p53 protein by yeast two-hybrid system]
Meiyun Zheng1, Miaojun Li1, Guoying Shen1
1College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China.
Objective:
To construct a mouse single-chain fragment variable (scFv) antibody library specific to human P53 by overlapping PCR method and screen the single chain antibodies against human P53 protein with the yeast two-hybrid (Y2H) system.
Methods:
The bait vector pGBKT-p53 expressing P53 protein in yeast AH109 cells was constructed by means of genetic engineering method. The total RNA which was extracted from the P53-immunized mouse spleen tissue was used to synthesize the single chain V(H)-linker-V(L) fragment by reverse transcription-PCR and overlapping PCR. And then the V(H)-linker-V(L) fragment constructed on the vector pGADT7 was transformed into the AH109 cells containing the bait vector. The positive clones were screened on the nutrition auxotrophic media. The characteristics of scFv were verified by immunocytochemistry.
Results:
The bait vector pGBKT7-p53 was constructed successfully and transformed into AH109 cells. It had no self-activation in the yeast cells and no toxicity to the host. The library of V(H)-linker-V(L) was successfully obtained. The captured vector harboring V(H)-linker-V(L) fragments was constructed. We screened out the human P53 scFvs by the Y2H system. And scFv1/2/3 was proved to have a good affinity for human P53 protein, so it could be used for the identification of P53 protein in cells and tissues.
Conclusion:
We obtained human P53 scFvs with a good affinity for human P53 protein by Y2H system, which provided a new way for screening scFv.
Insights
Researchers developed a mouse single-chain variable fragment (scFv) antibody library targeting human P53 protein. This library, screened using the yeast two-hybrid system, yielded scFvs with high affinity for P53 identification.
Area of Science:
- Immunotechnology
- Molecular Biology
- Biochemistry
Background:
- The tumor suppressor protein P53 is crucial in cellular responses to DNA damage.
- Developing specific antibodies for P53 is vital for diagnostic and research applications.
- Existing methods for antibody generation can be time-consuming and resource-intensive.
Purpose of the Study:
- To construct a mouse single-chain variable fragment (scFv) antibody library against human P53.
- To screen and identify scFvs with high affinity for human P53 using the yeast two-hybrid (Y2H) system.
- To establish a novel method for scFv screening.
Main Methods:
- Construction of a P53-expressing bait vector (pGBKT-p53) in yeast AH109 cells.
- Synthesis of single-chain V(H)-linker-V(L) fragments from P53-immunized mouse spleen RNA via RT-PCR and overlapping PCR.
- Transformation of scFv fragments into yeast and screening of positive clones using the Y2H system.
Main Results:
- Successful construction and validation of the P53 bait vector in yeast without self-activation or toxicity.
- Generation of a functional scFv library targeting human P53.
- Identification of three scFvs (scFv1/2/3) demonstrating good affinity for human P53, suitable for protein identification.
Conclusions:
- Successfully generated human P53-specific scFvs with high affinity using the Y2H system.
- The developed scFv library and screening method offer a valuable tool for P53 research.
- This approach provides a new avenue for efficient scFv discovery and application.
