Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition

Abstract

Highlights d Redesign of molecular recognition specificity, such as DNA binding, is difficult d Rounds of mutation and selection during protein engineering destabilize the scaffold d Stabilization of the protein scaffold prior to engineering increases success d These points are illustrated via a series of three protein engineering experiments

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method

“…PROSS designs were shown to improve thermal stability as well as functional expression yields in many cases without disrupting molecular activity [16,17,19,[21][22][23]47].…”

“…PROSS variants exhibited large gains in thermal stability of 15-20℃ and orders of magnitude of improvement in heterologous expression levels while maintaining wild type activity levels. To enable its widespread adoption, PROSS was implemented as an online server (http://pross.weizmann.ac.il/) that has attracted >1,500 academic users so far and led to several publications from other groups that demonstrated substantially stabilized protein variants [19,20,[23][24][25]. Though these results are encouraging, they do not represent a systematic analysis of PROSS's scope, and it is likely that experiments that indicated small improvements or a deterioration in stability following design would typically not be published.…”

Computational design and experimental characterisation of a stable human heparanase variant

“…In previous applications of PROSS, it was noted that large positively charged patches, which could promote aggregation in the nucleic acid rich micro environment, 37 were eliminated or reduced. 60,61 Here, in the case of HPSE, the electrostatic potential surface of HPSE WT shows two large positive patches around the active site and the b-sandwich domain (Fig. 3B).…”

Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies

“…Third, the produced variants are screened for activity in in vitro compartmentalised (IVC) systems: independent aqueous droplets with coupled transcription and translation capabilities and which carry DNA encoding both the MN and its target sequence. Multiple rounds of screening and selection are then performed, with increasingly stringent criteria: time for synthesis, time for cleavage, and temperature resistance [ 6 , 18 ]. Finally, this process can proceed iteratively to modify multiple contact modules or move on to validation of activity via expression and cleavage in bacteria.…”

“… Workflow for reprogramming the specificity of a meganuclease (MN) [ 6 , 18 ]. In the first stage, the target sequence must meet two conditions: (1) contain a central four recognition motif that matches the meganuclease to be modified and (2) have a minimal number of mismatches as compared to its original recognition sequence.…”