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A versatile tool in controlling aggregation and Ag nanoparticles assisted in vitro folding of thermally denatured
Preeti Gupta1, Ritu Verma1, Anita Kamra Verma2
1Molecular Biophysics Lab, Amity Institute of Biotechnology, Amity University, Sector 125, Noida, Uttar Pradesh, 201313, India.
Background:
Proteins have tendency to form inactive aggregates at higher temperatures due to thermal instability. Maintenance of thermal stability is essential to gain the protein in sufficient quantity and biologically active form during their commercial production.
Methods:
BL21-DE3 Rosetta E. coli cells which contains plasmid pET43.1a vector was used for producing zDHFR protein commercially. The purification of N-terminal Histidine tagged zDHFR was performed by Immobilized Metal Ion chromatography (IMAC). Investigations were performed in existence and non existence of Silver nanoparticles (AgNPs). The inactivation kinetics of zDHFR in existence and non existence of AgNPs were monitored over a range of 40-80 °C as monitored by UV-Visible absorption spectroscopy.
Results:
The protein completely lost its activity at 55 °C. Kinetics of inactivated zDHFR follows first order model in presence and absence of AgNPs. Decrease in rate constant (k) values at respective temperatures depicts that AgNPs contribute in the thermostability of the protein. AgNPs also assists in regaining the activity of zDHFR protein.
Conclusions:
AgNPs helps in maintaining thermostability and reducing the aggregation propensity of zDHFR protein.
General Significance:
Result explains that AgNPs are recommended as a valuable system in enhancing the industrial production of biologically active zDHFR protein which is an important component in folate cycle and essential for survival of cells and prevents the protein from being aggregated.
