scholarly journals A novel approach for the purification of aggregation prone proteins

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260143
Author(s):  
Austin Royster ◽  
Sheema Mir ◽  
Mohammad Ayoub Mir

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

2019 ◽  
Vol 294 (13) ◽  
pp. 5023-5037 ◽  
Author(s):  
Subbiah Jeeva ◽  
Sheema Mir ◽  
Adrain Velasquez ◽  
Jacquelyn Ragan ◽  
Aljona Leka ◽  
...  

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 115 ◽  
Author(s):  
Touraj Aligholipour Farzani ◽  
Katalin Földes ◽  
Koray Ergünay ◽  
Hakan Gurdal ◽  
Aliye Bastug ◽  
...  

Development of new vaccine platforms against viral diseases is considered urgent. In recent years, mRNA constructs have attracted great interest in this field due to unique advantages over conventional gene transfer platforms. In the present study, we developed a new naked conventional mRNA vaccine expressing the non-optimized small (S) segment of the Ank-2 strain of Crimean-Congo Hemorrhagic Fever virus (CCHFV). We then analyzed its single and booster dose immunogenicity and protection potential in the challenge assay in two mice models, including IFNα/β/γR−/− and C57BL/6. The results obtained from the immunological assays, namely IL-4 and IFN-gamma ELISPOT, intracellular IFN-gamma staining, in-house sandwich ELISA, and survival data, demonstrated that our construct elicited the production of anti-nucleocapsid (N) specific immune responses in both mice models. A 100% protection rate was only obtained in the booster dose group of IFNα/β/γR−/− mice, indicating that this platform needs further optimization in future studies. In conclusion, we assessed a novel approach in CCHFV vaccination by introducing a conventional mRNA platform which can be considered in future experiments as an efficient and safe way to battle this disease.


PLoS ONE ◽  
2017 ◽  
Vol 12 (9) ◽  
pp. e0184935 ◽  
Author(s):  
Subbiah Jeeva ◽  
Sean Pador ◽  
Brittany Voss ◽  
Safder Saieed Ganaie ◽  
Mohammad Ayoub Mir

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