Development and fabrication of disease resistance protein in recombinant Escherichia coli

Abstract

Cyanobacteria and Spirulina produce C-phycocyanin (CPC), a water soluble protein associated pigment, which is extensively used in food and pharmaceutical industries. Other therapeutic proteins might exist in microalgal cells, of which there is limited knowledge. Such proteins/peptides with antibiotic properties are crucial due to the emergence of multi-drug resistant pathogens. In addition, the native expression levels of such disease resistant proteins are low, hindering further investigation. Thus, screening and overexpression of such novel proteins is urgent and important. In this study, a protein which was identified as a putative disease resistance protein (DRP) in the mixture of Spirulina product has been explored for the first time. To improve protein expression, DRP was cloned in the pET system, co-transformed with pRARE plasmid for codon optimization and was significantly overexpressed in E. coli BL21(DE3) under induction with isopropyl-β-d-1-thiogalactopyranoside (IPTG). Furthermore, soluble DRP exhibited intense antimicrobial activity against predominant pathogens, and an inhibition zone of 1.59 to 1.74 cm was obtained for E. coli. At a concentration 4 mg/mL, DRP significantly elevated the growth of L. rhamnosus ZY up to twofold showing probable prebiotic activities. Moreover, DRP showed potential as an effective antioxidant, and the scavenging ability for ROS was in the order of hydroxyl > DPPH > superoxide radicals. A putative disease resistance protein (DRP) has been identified, sequenced, cloned and over-expressed in E. coli as a functional protein. Thus expressed DRP showed potential anti-microbial and antioxidant properties, with promising therapeutic applications.

Keywords

Disease resistance protein / Recombinant technology / Rare codon / Antibacterial / Antioxidant

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Funding

Ministry of Science and Technology, Taiwan(MOST 108-2218-E-006-006)