Design of protein with new function from existing protein scaffold

Abstract

Diverse functions of proteins have been evolved through the complex gene arrangement and further fine-tuning processes for a long time period. It is thought that these processes are accomplished by reusing or retaining limited protein progenitor scaffold. As a result, proteins having distinctly different physiological functions share the same structural scaffold and these are grouped into the superfamily enzymes. Though superfamily enzymes have the low sequence similarity, they share the similar protein scaffold and catalytic mechanism. Cyclic amidohydrolases and penicillin binding $proteins/\beta$ -lactamases are typical superfamily enzymes. The evolutionary relationship and common properties of superfamily proteins give the hint to the design of proteins with desired functions. In this experiment, new catalytic activity was designed and evolved from existing protein scaffold based on this information about superfamily enzymes. Target enzymes were TEM-1 $\beta$ -lactamase and penicillin binding protein 5 having similar mechanistic and structural properties. To create $\beta$ -lactamase activity from penicillin binding protein scaffold, the combinatorial approach of SIAFE and directed evolution was carried out. By using SIAFE approach, the functional elements for enhancing of substrate binding and introducing catalytic residues were incorporated into PBP5 scaffold in a random and combinatorial way. It was further optimized through three rounds of directed evolution by mimicking the natural protein evolution. Through in vitro evolutionary processes, finally evTEM variant having $\beta$ -lactamase activity was found. It has biased 36 amino acids mutations throughout the entire gene segment, mostly in the substituted loop regions as expected. The designed and incorporated functional elements were conserved during the extensive evolutionary processes and essential for evTEM to hydrolyze cefotaxime. Though its structural instability, evTEM showed distinct in vivo an...