Construction of Chimera Prolyl Endopeptidases to Determine Role of Domains in Substrate Size Specificity

Abstract

Eurygaster integriceps Puton (commonly known as the Sunn pest) is a member of the family Scutelleridae. It is a major pest of wheat and is commonly found in Middle East, northern Africa, Russia, and Eastern Europe (Critchley, 1998). The Sunn pest secretes a proteolytic enzyme, a prolyl endopeptidase (spPEP) into the wheat grains with subsequent degradation of gluten (Darkoh et al., 2010), the major protein in wheat. The spPEP is a unique prolyl endopeptidase in the family of S9 serine proteases because it recognizes large proteins (gluten) as substrates ranging from 30kDa to greater than 140kDa. All other prolyl endopeptidases are purported to bind only peptides shorter than 30 amino acids in length. PEPs consist of two conserved domains, a hydrolase domain which includes the catalytic triad (Ser554, Asp641, and His680) and a seven bladed β‐propeller domain (Juhász et al., 2006). To understand the role with which the two domains have on substrate specificity, chimeras were constructed in which domains were swapped between the sunn pest prolyl endopeptidase (spPEP), mammalian prolyl endopeptidase (hPEP), and the bacteria prolyl endopeptidase (bPEP). Forward and reverse primers identical within the hinge region of either spPEP, hPEP, or bPEP connecting the two domains were used in polymerase chain reaction (PCR) to amplify the vector and the respective domain of each PEP. Ligation independent cloning (LIC) was used to clone the insert DNA into the LIC vector (pNYCOMPS‐LIC‐ccdB‐FH10T+(N‐term) expression vector). The product chimeras include spPEP hydrolase/hPEP β‐propeller, spPEP hydrolase/bPEP β‐propeller, hPEP hydrolase/spPEP β‐propeller, hPEP hydrolase/bPEP β‐propeller, bPEP hydrolase/spPEP β‐propeller, and bPEP hydrolase/hPEP β‐propeller. Each of the chimeras were expressed in BL21 (DE3) pTFe. The cell lysates were used to determine the enzyme activity for each of the chimera using Gly‐Pro‐p‐nitroanalide (GPpNA) as substrate.Support or Funding InformationOffice of Research and Sponsored Programs, Stephen F. Austin State University, and Ed & Gowen Cole, Nacogdoches. Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.