An Inducible Expression System for Recombinant Sca Proteins with an Autotransporter Domain from Orientia Tsutsugamushi in Escherichia coli.

Abstract

Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative bacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization of the Ot strain into host cells and are suitable resources for vaccine development and tools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter (AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be promising candidates for vaccine development. These proteins are typically large and contain inhibitory domains; therefore, recombinant proteins without such domains have been evaluated for this purpose. However, the expression for recombinant Sca proteins containing the AT domain, which might largely affect their protective role against scrub typhus, has not been analyzed and optimized. In this study, we optimized expression and purification conditions for individual Ot Sca protein fragments [ScaA (27-1461), ScaC (257-526), ScaD (26-998), and ScaE (35-760)] harboring the AT domain. To this end, we subcloned sequences of codon-optimized DNA encoding Sca protein fragments into the Escherichia coli expression vector. In addition, the expression condition for individual Sca fragments was optimized, and the proteins were purified using one-step Ni-NTA column method. The purified fractions were re-folded by serial dilution method, followed by BCA quantification and densitometric analysis to estimate the yield and purity of proteins. We prepared platforms for expression of recombinant Sca protein fragments [ScaA (27-1461), ScaC (257-526), ScaD (26-998), and ScaE (35-760)] containing an AT domain without the signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with >70% of purity was ScaC (257-576), ScaE (35-760), ScaD (26-998), and ScaA (27-1461) in order. Our results could be used to develop Sca AT-domain based vaccines and tools for scrub typhus diagnosis with rapid and cost-effective ways.