Understanding GST-Survivin T34A AffiVECTOR: A Key Tool in Cancer Research

GST-Survivin T34A AffiVECTOR is a recombinant protein construct used in cancer research to study the role of survivin, a protein that inhibits apoptosis and is overexpressed in many cancers. This article provides an overview of the GST-Survivin T34A AffiVECTOR, its construction, applications, and significance in the field of oncology.

Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays a crucial role in inhibiting programmed cell death and regulating cell division. Its overexpression in cancer cells makes it a target for therapeutic research. GST (Glutathione S-transferase) fusion proteins, such as GST-Survivin T34A, are widely used to study protein-protein interactions and protein functions. The T34A mutation is a specific point mutation where threonine (T) at position 34 is replaced by alanine (A), which is known to affect the phosphorylation status and function of survivin.

Background on Survivin

Survivin is a 16.5 kDa protein encoded by the BIRC5 gene and is unique among IAP family members due to its dual role in inhibiting apoptosis and regulating mitosis. It is scarcely expressed in most differentiated adult tissues but is highly expressed in embryonic tissues and various human cancers, making it an attractive target for cancer therapy. The phosphorylation of survivin at threonine 34 by the p34cdc2-cyclin B1 complex is critical for its anti-apoptotic function.

Construction of GST-Survivin T34A AffiVECTOR

The GST-Survivin T34A construct is created by cloning the survivin gene with the T34A mutation into a vector that includes the GST tag. This allows for easy purification and detection. The steps involved include:

  • Gene Cloning: The survivin gene with the T34A mutation is amplified using PCR and cloned into a suitable expression vector containing the GST tag.
    • Primers Design: Primers are designed to specifically amplify the survivin gene and introduce the T34A mutation.
    • PCR Amplification: The gene is amplified using high-fidelity DNA polymerase to ensure accuracy.
    • Cloning into Vector: The amplified gene is inserted into the GST-tagged vector using restriction enzymes and ligase.
  • Transformation: The recombinant vector is introduced into E. coli cells for expression.
    • Competent Cells Preparation: E. coli cells are made competent to take up foreign DNA.
    • Transformation Procedure: The recombinant plasmid is introduced into the cells via heat shock or electroporation.
  • Protein Expression: Induction of protein expression in E. coli using IPTG.
    • Culture Growth: Transformed E. coli are grown to the log phase.
    • Induction: Protein expression is induced by adding IPTG to the culture.
  • Protein Purification: The expressed GST-Survivin T34A protein is purified using glutathione affinity chromatography.
    • Cell Lysis: Bacterial cells are lysed to release the protein.
    • Affinity Chromatography: The lysate is passed through a glutathione-Sepharose column to purify the GST-tagged protein.

Analytical Techniques

  • Western Blotting: Used to confirm the expression and purity of the GST-Survivin T34A protein.
    • SDS-PAGE: Proteins are separated based on their size.
    • Blotting: Proteins are transferred onto a membrane and detected using specific antibodies.
  • Mass Spectrometry: Confirms the molecular weight and identity of the purified protein.
    • Sample Preparation: The protein is digested with a protease such as trypsin.
    • MS Analysis: The resulting peptides are analyzed by mass spectrometry to confirm the protein's identity.
  • Functional Assays: Assess the impact of the T34A mutation on survivin’s function in apoptosis inhibition and cell division.
    • Apoptosis Assays: Cells expressing GST-Survivin T34A are tested for resistance to apoptotic stimuli.
    • Cell Cycle Analysis: Flow cytometry is used to analyze the cell cycle distribution of cells expressing GST-Survivin T34A.


The GST-Survivin T34A AffiVECTOR was successfully constructed and expressed in E. coli. Purification yielded a high-quality protein suitable for downstream applications. Western blotting confirmed the presence of the GST-Survivin T34A protein at the expected molecular weight. Mass spectrometry analysis verified the identity of the purified protein. Functional assays revealed that the T34A mutation significantly affects survivin’s ability to inhibit apoptosis, which is consistent with previous findings.


The GST-Survivin T34A AffiVECTOR provides a valuable tool for studying the biological functions of survivin and its role in cancer. The T34A mutation specifically allows researchers to dissect the importance of phosphorylation at this site. This construct can be used in various assays to screen for potential anti-cancer drugs targeting survivin. The ability to easily purify and detect the GST-tagged protein facilitates biochemical and structural studies, enhancing our understanding of survivin's interactions and functions.

Applications in Cancer Research

  • Drug Screening: GST-Survivin T34A can be used in high-throughput screening to identify compounds that affect survivin function.
  • Protein-Protein Interaction Studies: The GST tag allows for pull-down assays to identify interacting partners of survivin.
  • Structural Analysis: Crystallography and other structural techniques can be applied to study the three-dimensional structure of survivin and its complexes.
  • Functional Studies: Investigating the effects of survivin mutations on cell cycle regulation and apoptosis.

GST-Survivin T34A AffiVECTOR is a powerful research tool that aids in understanding the molecular mechanisms of survivin in cancer. Its ability to be easily purified and detected, combined with the functional insights it provides, makes it indispensable in cancer research. The T34A mutation offers a unique opportunity to study the role of phosphorylation in survivin’s function, paving the way for potential therapeutic interventions.

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