Characterization And Optimization Of Recombinant Gm-Csf Protein: Expression And Functional Analysis
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Abstract
Background: Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) emerges as a key influencer, wielding its impact over haematopoiesis and immune modulation. It can address an array of intricate physiological and pathological scenarios.
Objective: The present study is aimed to characterize and optimize the recombinant GM-CSF protein and to carry out its expression and functional analysis.
Methods: GS115 (Pichia pastoris) strain was double digested with restriction enzymes 5’AOX1 and 3’AOX1α and ligated to the pPICZα vector. The positive clones were screened using PCR, mobility shift, Kex2 signal cleavage and restriction digestion. Further, the Pichia strain with pPICZα A was transformed into yeast cells, and its expression was studied using Spectroscopy and SDS-PAGE. In addition, the resulting Protein was purified using reverse-phase column chromatography and functional characterization was performed using a Pichia pastoris HCP kit.
Results: The GM-CSF protein was successfully transformed into yeast cells, andtheSDS-PAGE profile confirmed the presence of GM-CSF in the expression system. A purified form of recombinant GM-CSF protein was obtained using HPLC, and the obtained chromatograms of both reference and test samples were comparable. Further, from the functional analysis, about9.67 ppm of the functional hematopoietic cell phosphatase (HCP) was observed in the purified GM-CSF sample.
Conclusion: The Recombinant GM-CSF has been successfully prepared and confirmed for their expression and functional characteristics. The developed Protein can regulate production, cell differentiation and granulocytic functions.
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