Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum.

Protein production relies on time-consuming genetic engineering and expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of protein biosynthesis by processing transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete The OPEL-like I06 protein of can stimulate host antibody responses, similar to the OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast protein synthesis. CFPS simultaneously generated multiple proteins of and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen. Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete , which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.