1. [Expression and purification of FLT3 and FLT3-ITD mutated proteins in HEK293T cells].
- Author
-
Yang F, Du J, Wu J, Yuan P, Su Q, Han R, Qiu Q, Xue S, Liu S, and Sun Z
- Subjects
- Blotting, Western, Bone Marrow Cells chemistry, Bone Marrow Cells metabolism, HEK293 Cells, Humans, Leukemia, Myeloid, Acute genetics, fms-Like Tyrosine Kinase 3 isolation & purification, Gene Expression, Leukemia, Myeloid, Acute enzymology, Mutation, fms-Like Tyrosine Kinase 3 genetics, fms-Like Tyrosine Kinase 3 metabolism
- Abstract
Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 (FLT3) gene and FLT3-internal tandem duplication (FLT3-ITD) mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-ITD were amplified from bone marrow cells of healthy individuals and FLT3-ITD-mutated acute myeloid leukemia (AML) patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD plasmids were transfected in HEK293T cells by Polyjet reagent, and the recombinant proteins were purified by immunoprecipitation and competing elution methods. Results FLT3wt and FLT3-ITD-mutated DNA sequences were successfully cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD mutated proteins were successfully expressed and purified in HEK293T cells as verified by Western blotting and sliver staining. Conclusion FLT3wt and FLT3-ITD expression vectors were successfully constructed, and purified proteins were successfully obtained from HEK293T cells.
- Published
- 2018