Purification,identification And Structural Study Of The Royal Jelly Proteins MRJP1-3

Major Royal Jelly Proteins(MRJPs)are the important functional protein family in royal jelly.Besides serving as nutrient to queens and larvae,MRJPs also regulate the caste differentiation of bees.The function of biomacromolecule mainly depends on its structure,but the research about natural secondary structure of MRJPs has not been reported yet.This paper focuses on the purification and secondary structure of MRJP1 monomer,MRJP1 oligomer,MRJP2 and MRJP3 from royal jelly(Apis mellifera).The main results are as follows:First,MRJP1 monomer and MRJP1 oligomer were separated and purified through anion-exchange chromatography combined with gel filtration chromatography using ToyoScreen GigaCap Q-650M column and Superdex 200 10/300 GL column.MRJP2 was separated and purified with cation-exchange chromatography and anion-exchange chromatography using ToyoScreen GigaCap S-650M column and ToyoScreen GigaCap Q-650M column.MRJP3 was purified only with cation-exchange chromatography using ToyoScreen GigaCap S-650M column.Next,liquid chromatography with tandem mass spectrometric detection methods(LC-MS/MS)was used to identify MRJP1 monomer,MRJP1 oligomer,MRJP2 and MRJP3,respectively.The sequence coverages of these proteins were 44.01%,53.47%,49.34%and 39.35%separately.Finally,secondary structures of MRJP1 polymer,MRJP1 monomer,MRJP2 and MRJP3 were detected by synchrotron radiation circular dichroism technology(SRCD).The results show that the proportion of secondary structure in MRJP1 monomer was that 3 strand accounted for 35%,unordered accounted for 34%,? turn accounted for 21%,a helix accounted for 10%at room temperature.In MRJP1 oligomer,? strand accounted for 30%,unordered accounted for 29%,?turn accounted for 23%,a helix accounted for 18%.In MRJP2,? strand accounted for 30%,unordered accounted for 27%,? turn accounted for 23%,a helix accounted for 19%.In MRJP3,unordered accounted for 43%,a helix accounted for 27%,? strand accounted for 15%,? turn accounted for 15%.The results reveal that the temperature affected the secondary structures of them.Denaturation temperature of MRJP1 monomer was 35?,MRJP1 oligomer was 45?,MRJP2 was 45?,and MRJP3 was 55?.The results of this paper obtained a simple and efficient method for the purification of MRJP1 monomer,MRJP1 oligomer,MRJP2 and MRJP3.In addition,information about the structures of MRJPs may provide theoretical basis to understand structural changes of royal jelly proteins during storage.