Optimized Solubilization and Purification of Recombinant Teriparatide Fusion Protein Expressed in E. coli
Author(s):
Message:
Abstract:
Aims
There are several cell disruption methods for intracellular protein extraction. The aim of this study was to select the best approach for recombinant teriparatide fusion protein extraction from E. coli and achieve the best purification conditions.
Materials & Methods
In this experimental research, bacterial cells were disrupted by different methods such as sonication in different cycles, grinding with liquid nitrogen in two different cell culture volumes, and homogenization at two different pressures. The supernatant and pellet samples were run on sodium dodecyl sulphate gel. All the cell lysates were cultured on LB agar medium and stained with Gram staining method. The Ni2+ affinity chromatography of recombinant teriparatide fusion protein was done under denaturing and non-denaturing conditions, using pH and imidazole concentration gradient, respectively. All samples were taken on sodium dodecyl sulphate-polyacrylamide gel and the amount of purified protein was calculated by Micro-Bradford assay.
Findings
In the 20 and 25 cycles, a large part of the fusion protein led to protein solubilization. In the method of grinding with liquid nitrogen, proteins were more likely to enter the sediment part. The cell disruption was complete in a chemical method. The cell disruption under 50bar homogenization was more than that of 15bar. In chemical degradation and sonication, a large amount of fusion protein led to protein solubilization. In non-denaturing conditions, no recombinant fusion protein was removed from the column with the isolation buffer, but in the denaturing conditions, a large amount of proteins was purified.
Conclusion
The combined method of chemical degradation and sonication leads to approximately 97.7% of protein solubilization, and the purification in denaturing condition has also the suitable result in contrast to non-denaturing condition.
Language:
Persian
Published:
Modares Journal of Biotechnology, Volume:10 Issue:1, 2019
Pages:
1 - 7
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