Rational design of recombinant proteins to increase the stability and solubility has been a goal of protein technology and has significant implications in biotechnology research
Rational design of recombinant proteins to increase the stability and solubility has been a goal of protein technology and has significant implications in biotechnology research. Poorly soluble enzymes increase the cost and have a negative impact on the feasibility of the process due to need for larger reaction volumes, longer incubation times, and more restricted reaction conditions.
Tobacco Etch Virus protease is a protease with stringent specificity and efficiency that is widespread used for the cleavage of fusion proteins. However, a major limitation of this enzyme is its relatively poor solubility (generally; 1 mg/mL). Following a rational process to determine stabilizing and solubilizing mutations of TEV protease using online tools such as PoPMuSiC and Eris servers, five mutations include N23F, N23L, Q74L, Q74V and Q74I were suggested for further studies. Best models of wild type and mutations made using Modeller 9.13 software and quality of them checked by PORCHECK and Verify3D servers. MD simulations performed to evaluate the dynamic behavior of wild type and mutant structures of TEV protease. Totally, after analysis of various properties relative to stability and solubility, N23F mutant chose for in vitro studies. This mutation has increased the solubility 1.5 times and improved melting temperature almost 1 ? C than wild type although; its activity has decreased almost 5.5 times than wild type. In this study, we have proposed a general method that can be used to analysis the stability and solubility of other similar proteins.
