Essay: Biochemical and biophysical characterization of Zebrafish DHFR

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There have been many detailed studies on the folding of small monomeric proteins (ref…………………………………………..). Even there are many reports on DHFR protein of different origins (ref…………………………………………….). However, equilibrium unfolding and refolding study of zebrafish DHFR has not been reported till date. In the present work, we have established the equilibrium folding scheme of zDHFR after obtaining thermodynamic parameters of the protein. It undergoes a spontaneous folding process without any molecular participation and refolds completely when denatured in presence of GdnHCl and urea. In order to perform the thermodynamic studies of the folding of zDHFR, a reversible, path independent folding of ZDHFR had to be demonstrated.
Overexpression in zDHFR
In the present study, pET 43.1a vector bearing the zebrafish DHFR gene under the control of T7 promoter was used for over-expression of zDHFR in E. coli expression system. It has T7 RNA polymerase machinery under the control of Lac promoter. The overexpression of zDHFR protein was carried out by adding non-hydrolysable analogue of lactose, IPTG which typically acts as chemical inducer. The conditions for overexpression of zDHFR were optimized w.r.t. its concentration (Figure 4.3 a) and duration of incubation (Figure 4.3 b). The recombinant protein over expressed in the host cellular system should be sufficiently active at the time of induction. Thus, cell concentration before induction plays a crucial role for over-production of recombinant proteins (OD600 should be around 0.8- 1.0). A good level of expression in zDHFR protein was obtained in E. coli cells when induction was carried out in the mid-exponential phase. Over-expression was performed using uninduced sample as control.
Enhancement of over expression by osmolytes
Most of the over expressed recombinant proteins in bacterial cell cannot reach to a correct conformation and undergo proteolytic degradation or associate with each other and often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. Hence, there is an ever-growing interest in developing strategies to avoid protein aggregation or to enhance protein refolding yields. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or chaperones. Osmolytes are naturally occurring organic compounds affecting osmosis and they protect organisms from stress induced by osmotic pressure. It represents diverse chemical categories including amino acids, methylamines, and polyols. Due to increased concentrations of osmolytes, organisms may undergo some conformational changes in cellular proteins. Osmolytes shift equilibrium toward natively-folded conformations by increasing the free energy of the unfolded state. Osmolytes mainly affect the protein backbone. This balance between osmolyte’backbone interactions and amino acid side chain’solvent interactions decides protein folding process.
Abnormal cell volume regulation significantly contributes to the pathophysiology of several disorders, and cells respond to these changes by importing, exporting, or synthesizing osmolytes to maintain volume homeostasis. In recent years, it has become quite evident that cells regulate many biological processes such as protein folding, protein disaggregation, and protein’protein interactions via accumulation of specific osmolytes. Many genetic diseases are attributed to the problems associated with protein misfolding/aggregation, and it …

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