As part of JST Strategic Basic Research Programs, a group of researchers led by YOMO Tetsuya (Professor, Graduate School of Information Science, Osaka University) and MATSUURA Tomoaki (Associate Professor, Graduate School of Engineering, Osaka University) succeeded in evolving membrane proteins using artificial cells in vitro.
Methods to change or improve the properties of proteins through replication in labs in accordance with Darwin's theory of evolution by mutation and natural selection was developed in the 90s. Since then, such methods have attained great achievements in the development of medicines. However, membrane proteins are generally insoluble and hard to express in different cells. Therefore, despite their biological and pharmaceutical importance, artificial modification has not been achieved using membrane proteins.
This group found that membrane proteins were integrated into the artificial membranes by synthesizing membrane proteins from genes in cell-sized artificial lipid bilayer membranes (liposomes) containing substances necessary for synthesizing proteins. Furthermore, this group confirmed the activity of membrane proteins to integrate substances into the membrane from outside the artificial cells.
Moreover, this group succeeded in selecting genes to encode highly functional membrane proteins from a group of mutation-induced genes. Using this technique, they succeeded in artificially increasing the function of α hemolysin, a membrane protein, about 30 times relative to the wild type. This high performance α hemolysin is expected to increase the performance of DNA sequence analyzer using α hemolysin. Moreover, this method called liposome display can be applied to a variety of membrane proteins. Thanks to this method, it will become possible to make "convenient" membrane proteins with different or high functions. Thus, it will contribute to the development of high performance bio-sensors as well as the development of new drugs to combine with membrane proteins to interfere with function.

Abstract

In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g., antibodies, enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method called liposome display that can evolve the properties of membrane proteins entirely in vitro. This method, which involves in vitro protein synthesis inside liposomes, which are cell-sized phospholipid vesicles, was applied to the pore-forming activity of α-hemolysin, a membrane protein derived from Staphylococcus aureus. The obtained α-hemolysin mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the WT. Given the ability to synthesize various membrane proteins and modify protein synthesis and functional screening conditions, this method will allow for the rapid and efficient evolution of a wide range of membrane proteins.

Related links :

• Yomo Research Group, Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University
• Watabnabe Lab. Bioenvironmental Science, Osaka University