Expression analysis of the nucleocytoplasmic lectin ‘Orysata’ from rice in Pichia pastoris

Abstract

The Oryza sativa lectin, abbreviated Orysata, is a mannose‐specific, jacalin‐related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin‐related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomycesα‐factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N‐glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high‐mannose structures, Man9–11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high‐mannose as well as with more complex N‐glycan structures. Orysata has potent anti‐human immunodeficiency virus and anti‐respiratory syncytial virus activity in cell culture compared with other jacalin‐related lectins.

Characterization of a type D1A EUL-related lectin from rice expressed in Pichia pastoris

“…Over the last 5 years at least eight (Jiang et al, 2010) types of lectin domain have been reported in rice, most of which can be considered as stress-inducible lectins. The jacalin-related lectin Orysata and the EUL-related lectin OrysaEULS2 have both been shown to exhibit specificity towards mannose (Al Atalah et al, 2011. This report yields evidence that at least OrysaEULD1A is directed towards galactosylated carbohydrates.…”

“…The full EULD1A sequence from rice as well as its individual EUL domains EULD1A domain 1 and EULD1A domain 2, have been successfully expressed in P. pastoris. On average, the yield obtained for the recombinant proteins can be considered low compared to the yield reported previously for some other plant lectins (Oliveira et al, 2008;Al Atalah et al, 2011). However a yield of 0.5-3 mg/l recombinant protein is comparable to the yield obtained for OrysaEULS2 (4-6 mg/l) (Al Atalah et al, 2012).…”

“…This vector also contains a polyhistidine tag, which allows detection of the recombinant proteins as well as purification by metal-affinity chromatography. After Pichia transformation, several colonies per construct were tested for EUL expression and the colonies that yielded the highest expression were selected to grow larger Pichia cultures following the procedure described by Al Atalah et al (2011), except that Pichia cells were grown and expression was induced at 22°C instead of 30°C.…”

“…These inducible lectins are often present at low but physiologically relevant concentrations in plant tissues. As they are expressed in the cytoplasm and/or nucleus of the plant cell (Al Atalah et al, 2011;Van Hove et al, 2011), they are referred to as nucleocytoplasmic lectins (Lannoo and Van Damme, 2010). Recently, the hypothesis has been put forward that inducible lectins play an important role in the stress physiology of the plant cell through proteincarbohydrate interactions taking place in the cytoplasm and the nucleus (Van Damme et al, 2004a,b, 2011Lannoo and Van Damme, 2010).…”

“…To overcome this problem, the OrysaEULD1A sequence was expressed in Pichia pastoris under the control of the alcohol oxidase 1 promoter (AOX1), known to induce high levels of protein after induction with methanol (Hartner and Glieder, 2006). In recent years, multiple nucleocytoplasmic lectins have been successfully expressed and purified from P. pastoris (Lannoo et al, 2007;Fouquaert et al, 2009b;Al Atalah et al, 2011;Van Hove et al, 2011;Al Atalah et al, 2012;Stefanowicz et al, 2012).…”