The Molecular Design, Soluble Expression And Characterization Of A Thermostable Chimeric Esterase

People can change protein molecular characteristics and do research more deeply according to the development of protein molecular design which is composed of de novo protein design and rational design based on natural proteins. Furthermore, the development of bioinformatics also gives great supports to protein molecular design.In this work, we design a protein named AAM6 which possesses the catalytic domain of mutant R526V and the cap domain of AFEST. R526V and AFEST are the proteins with low sequence homology. The data of MD and experiments are analysised. In addition, the research on two parts was performed. First, a recombinant protein AAM6 is getted which contains two domains from different proteins without sequence homology, but with the similar 3D structure. Second, research on substrate specificity ofα/βfold superfamily is carried out.In this work, the recombinant segments originate from the two thermophilic esterases (mutant R526V and AFEST). After the alignment of two proteins by VAST in NCBI and the analysis of linker regions of APE1547 and AFEST using LPC/CSU, the recombinant site is chosen and the model of AA is constructed by modeller.However, there are problems about AA in expression. We can not get soluble protein in supernatant. To solve this problem, we do a series of experiments such as changing hydrophobic residues and refolding the inclusion bodies, et al. Then, we get soluble protein AAM6 by using culture medium with only 1/4 amount of NaCl as normal.The docking and molecular dynamics simulation on AAM6 is carried out. We also investigate the molecular characteristics and substrate specificity on AAM6. The results show that AAM6 inherits the thermostability of its parents and even better than its parents. As hypothesized, AAM6 prefers the hydrolysis of short chain acyl esters in the subtrates specificity. However, the results of docking show that AAM6 has no substrate specificity about C2~C10. This result is not consistent with the experimental result. In order to get insight into the problem, we construct a model of AAM6 with His-tag and do molecular dynamic simulation on this model using gromacs. The result of simulation shows that the His-tag folds and blocks the possible entrance of AAM6. This result can be well consistent with experiments. With the increase of the temperature, the molecular becomes more flexible and the entrance may open more widely. However, the width of the entrance is not fixed, and the small substrates are esaier to get through the entrance and reach the catalytic center.The significance of our work can be summarized as follows:First, we can construct a recombinant protein by rational design when the X-ray structure has been studied. Second, the substrate specificity is not only restricted to the binding site, but also related to the entrance. More researches on this phenomenon will be significant to know more about the relationship between protein structure and substrate selectivity.