부산대학교 도서관

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (Os RGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of Os RGLP1 to study the role of glycosylation on Os RGLP1 protein stability and activity. Sequence analysis of Os RGLP1 homologs identified diverse N -glycosylation sequons, one of which was highly conserved. We therefore expressed Os RGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified Os RGLP1 showed it was expressed by S. cerevisiae in both N -glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP- Os RGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP- Os RGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for Os RGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that Os RGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N -glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of Os RGLP1 which paves the way for further characterization and use of this protein. • Oryza sativa Root Germin-like Protein 1 has a highly conserved N -glycosylation site. • Os RGLP1 has super oxide dismutase activity. • N -glycosylation of Os RGLP1 is not required for stability or activity. • Os RGLP1 can be efficiently expressed in K12 shuffle E. coli. [ABSTRACT FROM AUTHOR]