Optimization of Expression and Purification of some Model Histidine-tagged Recombinant Proteins: MiRGD, GNH, HNH, Firefly Luciferase and Human DT-Diaphorase

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Article Type:
Research/Original Article (دارای رتبه معتبر)
Abstract:
Higher rates of protein expression lead to the accumulation of proteins as inclusion bodies. In contrast, the expression of soluble proteins needs to be optimized to reduce the amount of accumulated proteins. Therefore, obtaining a high amount of the desired his- tag recombinant proteins always requires a balance between expression rate and accumulation. Inducer concentration, time, and temperature of induction are the main variable in the level of soluble protein or inclusion bodies expression. In summary, if the higher concentrations of the protein are toxic or tend to be aggregated inside cells, higher ODs, lower IPTG concentrations, and shorter expression times can be effective in formation of inclusion bodies. The standard purification process can also be optimized based on the Physicochemical properties, such that, in addition to increasing yield in the expression step, the activity and concentration of the desired protein is maintained in the purification step. In this study, we obtained the results of extraction of three different peptides MiRGD, GNH and HNH, and two proteins firefly mutant red-emitter luciferase, and human DT-diaphorase proteins by altering these variables on expression as well as purification conditions. Optimization of the protocols in Ni-NTA affinity chromatography of recombinant proteins with denaturants for MiRGD, GNH and HNH brought about with increase of purified protein concentrations from 0.21, 0.4, and 0.16 mg/ml in the initial attempt to 2.9, 3.4 and 2.6 mg/ml in the optimized experiment respectively. Non-denaturing optimization of purification for mutant luciferase and human diaphorase produced 4.1 and 2.6 mg/ml, respectively.
Language:
English
Published:
Biomacromolecular Journal, Volume:7 Issue: 3, Winter 2021
Pages:
93 to 102
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