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Archive > Volume 31 Issue 7 > 2024,31(7):669-674. DOI:10.3872/j.issn.1007-385X.2024.07.004 Prev Next
Basic Research
Expression and characterization of ENO1 protein and its associated active site deletion mutant proteins in a baculovirus expression vector system
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Abstract:
Objective: To express the glycolytic enzyme alpha-enolase (ENO1) and its three enzyme active site deletion mutant ENO1 proteins (ENO-M1, pFastBac-M2, ENO1-M3) using a baculovirus expression vector system (insect BEVS), laying the groundwork for the subsequent study of metabolic therapy for cervical cancers. Methods: Molecule cloning was used to insert optimized ENO1 gene sequence into the pFastBacTM1 vector to obtain the recombinant plasmid pFastBac-ENO1 with target genes. Three active sites essential for ENO1's glycolytic function were deleted, and the corresponding optimized sequences were inserted into the pFastBacTM1 vector to generate the recombinant plasmids with three active site deletion, namely pFastBac-M1, pFastBac-M2 and pFastBac-M3. Recombinant baculoviruses rBV-ENO1, rBV-M1, rBV-M2 and rBV-M3 were subsequently obtained through transposition and transfection. The expression and specificity of the target proteins were examined using WB assay. Results: Recombinant bacilli rBacmid-ENO1, rBacmid-M1, rBacmid-M2 and rBacmid-M3 were successfully amplified, obtaining a gene fragment of about 2 000 bp in size, which was consistent with the expected size. The insect BEVS could express the ENO1 protein and its recombinant proteins (ENO1-M1, ENO1-M2, ENO1-M3) with three enzyme active site deletions, each with a molecular weight of approximately 52 000, as expected. WB analysis confirmed that these proteins reacted with the specific His-tag antibody. Conclusion: The insect BEV successfully expresses the target protein and its proteins with enzyme active site deletions, namely ENO1-M1, ENO1-M2 and ENO1-M3. This protein's reactivity establishes the foundation for subsequent determination of the affinity of these proteins, and ENO1 monoclonal antibody.
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