The immunostimulatory effect of the recombinant apalbumin 1–major honeybee royal jelly protein–on TNFα release

Apalbumin1 (Apa1) is the major royal jelly (RJ) and honey glycoprotein having various biological properties. We have previously demonstrated that Apa1 is a regular component of honey and honeybee pollen and stimulates macrophages to release tumor necrosis factor α (TNFα). The recombinant Apa1 (rApa1) and its four recombinant protein fragments derived on the basis of partial tryptic products of Apa1 were prepared by heterologous expression in Escherichia coli BL21-CodonPlus(DE3)-RIL. l-Arginine at 50 mM concentration was used for improving the recombinant protein solubility. We report that the proteinous moiety of glycoprotein is responsible for stimulation of TNFα production by murine peritoneal macrophages. Moreover, we have shown that immunostimulatory effect is significantly increased after partial tryptic digestion of Apa1. It has been determined that recombinant N-terminal fragment of Apa1 is the most active elicitor of TNFα release in comparison to other three protein fragments of Apa1, as well as to the native Apa1 and rApa1.Furthermore, it was found that native honey was able to stimulate TNFα secretion from murine macrophages, whereas the deproteinized honey had no effect on the release of TNFα. This result suggests that immunostimulatory effect of honey is based on its RJ-protein content, primarily on its dominant protein Apa1.