Purification And Identification Of Major Royal Jelly Protein 1-3 And Changes In The Process Of Storage

Royal jelly(RJ)possesses numerous functional properties to benefit human health.Major royal jelly proteins(MRJPs)as the most characteristic component of royal jelly,has become the focus of current research interests.In this study,royal jelly collected from European honeybees(Apis mellifera)was researched.We aimed to propose a sequential processing strategy to isolate and purify MRJP 1-3 from royal jelly,and a high sensitivity SEC-HPLC was used to identify the purity of separated proteins for the first time.Meanwhile,SEC-HPLC was also used to detect protein changes in royal jelly under different storage conditions.The results are as follows:1.In this study,we establish a sequential process to purity the natural MRJP 1-3 by using three different chromatographic procedures: hydrophobic interaction chromatography,size-exclusion chromatography and cation exchange chromatography.In the first step,MRJP1 oligomers and monomer were isolated from crude soluble royal jelly protein by a hydrophobic interaction chromatography on Hitrap Butyl HP.In the second step,MRJP 1 oligomer 1,MRJP 1 oligomer 2 and MRJP 1 monomer were respectively purified by a size exclusion chromatography on HiLoad X16/60 Superdex 200 pg.Finally,MRJP2 and MRJP3 s were respectively isolated from the unbound proteins derived from the first step by a cation exchange chromatography on Toyopearl GigaCap S-650 M.2.Liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS)was carried out to identify the five purified individual MRJPs.The sequence coverages of the purified MRJP 1 oligomer 1,MRJP 1 oligomer 2,MRJP 1 monomer,MRJP 2 and MRJP 3s were 92.13%,76.62%,91.20%,87.39% and 64.71%,respectively.MRJP 1 oligomer 2 was first isolated and identified as oligomeric formation of MRJP 1 with molecular weight of 670 kDa.By using SEC-HPLC to detect the purity of each separated protein as follows: the purity of the purity of MRJP 1 oligomer 1 was 89.88%;MRJP 1 oligomer 2 was 67.79%;the purity of MRJP 1 monomer was 99.92%;the purity of MRJP 2 was 99.41%;the purity of MRJP 3s was 99.97%.3.Analytical ultracentrifugation(AUC)experiments were performed to determine the more accurate molecular weight(MW)of MRJP 1 oligomers,as 228 kDa MRJP 1 oligomer 1,408 kDa MRJP 1 oligomer 2 and 639 kDa MRJP 1 oligomer 3,respectively.All the MRJP 1 oligomers were made up of MRJP 1 monomer and Apisimin.We speculated that the MRJP 1oligomer 2 and 3 maybe were the dimer and trimer of MRJP 1 oligomer 1.According to the size-exclusion chromatography,Native-PAGE and LC-MS/MS analysis results,we considered that MRJP 1,the most abundant protein in royal jelly,exists predominantly as oligomers in RJ,and the amount of 228 kDa oligomer formation was higher,while the amount of MRJP 1 monomer was lower.4.Synchrotron radiation circular dichroism(SRCD)measurements were performed to detect the secondary structure of the five purified individual MRJPs.The results indicated that all the five purified proteins consisted predominantly of ?-sheets and random coil,while less of ?-Helix.5.When analyzing royal jelly protein by size-exclusion chromatography,nine representative peaks were obtained.Electrophoresis analysis,protease hydrolysis,indene three ketone colorimetric method,and automatic amino acid analysis were performed to identify the nine peaks fraction.As the results,the peaks 1-4 had been assign to MRJP 1 oligomer 2,MRJP 1 oligomer 1,MRJP3 s and mixture of MRJP2 and MRJP1 monomer in sequence,while the peaks 5-9 had been assign to free amino acids and small peptides.6.Size exclusion chromatography high performance liquid chromatography(SEC-HPLC)was used to detect protein changes in royal jelly under different storage conditions.Our results shows that the protein composition of fresh royal jelly had no change during storage six months at-18?,the protein composition of fresh royal jelly had a slow change stored after one month at 4?;the protein composition of fresh royal jelly had an obvious change stored within one month at room temperature.As storage time increased during room temperature and 4?,the proportion of 290 kDa MRJP 1 oligomer,670 kDa MRJP 1 oligomer and MRJP 1 monomer showed a trend for decreasing,while the emergence of new 700 kDa MRJP 1 oligomer and 440 kDa MRJP 1 oligomer showed a trend for increasing.Therefore,we speculate that MRJP 1 monomer maybe crosslinked with existing oligomers to form new oligomers as storage time increased.In addition,we found that if peak 1 area ratio less than 0.50,peak 2 area ratio more than 0.55 and peak N area ratio less than 0.02,the royal jelly sample detected by SEC-HPLC should be consider as fresh.So,based on the SEC-HPLC,we had set up a new method for the determination of the royal jelly freshness.