Major Royal Jelly Proteins Research Articles

Major royal jelly proteins prevents NAFLD by improving mitochondrial function and lipid accumulation through activating the AMPK / SIRT3 pathway in vitro.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic syndrome, whose main characteristics are excessive lipid accumulation and oxidative stress. Major royal jelly proteins (MRJPs) is a kind of water-soluble protein, which is abundant in royal jelly (RJ). The aim of this study was to evaluate the effect of MRJPs on lipid accumulation and oxidative stress of liver cells. Here, we first optimized the conditions for extracting MRJPs from RJ and identified the extraction effect and product by SDS-PAGE. Then, we used oleic acid (OA) of 1.0mM to induce hepatocytes for 24 hr to establish a stable cell models of lipid accumulation, and we found that pre-administration (24 hr) of MRJPs (0.2, 0.5, and 1.0g/L) could significantly reduce the lipid drop content and triglyceride level in the model cells, and simultaneously reduce the alanine aminotransferase and aspertate aminotransferase levels in the cell culture supernatant. In addition, pre-incubation (24 hr) with MRJPs (0.2, 0.5, and 1.0g/L) could restore superoxide dismutase (SOD) level and mitochondrial membrane potential as compared with OA group. Furthermore, MRJPs administration significantly upregulated the expression of Silent Information Regulator 2 Associated Protein 3, mitochondrial superoxide dismutase (SOD2), and cytochrome c oxidase subunit IV in OA-treated HepG2 cells. The study for the first time provides evidences on the lipid-lowering effect of MRJPs at the cellular level, which can further provide support for the development and application of polypeptide drugs in the future, and can also provide a choice for the prevention and treatment of liver metabolic diseases represented by NAFLD. PRACTICAL APPLICATION: Our study proved that MRJPs had substantial preventing effect on OA-induced lipid accumulation and mitochondrial dysfunction in HepG2 cells. This research can further provide theoretical support for the development and application of peptide drugs in the future. Besides, it can not only further broaden our understanding of NAFLD and other diseases, but also provide ideas for research on oxidative stress and lipid accumulation in the body.

Health Promoting Properties of Bee Royal Jelly: Food of the Queens.

Royal jelly (RJ) demand is growing every year and so is the market for functional foods in general. RJ is formed by different substances, mainly carbohydrates, proteins, and lipids, but also vitamins, minerals, and phenolic or volatile compounds in lower proportion. Major royal jelly proteins (MRJP) are, together with 10-hydroxy-2-decenoic acid (10-HDA), key substances of RJ due to their different biological properties. In particular, 10-HDA is a unique substance in this product. RJ has been historically employed as health enhancer and is still very relevant in China due to the traditional medicine and the apitherapy. Nowadays, it is mainly consumed as a functional food or is found in supplements and other formulations for its health-beneficial properties. Within these properites, anti-lipidemic, antioxidant, antiproliferative, antimicrobial, neuroprotective, anti-inflammatory, immunomodulatory, antiaging, and estrogenic activities have been reported for RJ or its specific components. This manuscript is aimed at reviewing the current knowledge on RJ components, their assessment in terms of authenticity, their biological activities, and related health applications.

A new design of bee cage for laboratory experiments: nutritional assessment of supplemental diets in honey bees (Apis mellifera)

We designed a cage that can maintain adult bees for 21 days with about 90% survival rate, in addition to facilitating the changing of sucrose syrup and beebread, and collection of live and dead bees without disturbing or releasing bees. The consumption rates of bee pollen diet and sucrose syrup for groups of 50–200 honey bees were recorded for 21 days after emergence. The bees consumed the bee pollen diet mainly at the age of 1–9 days. Regular consumption of sucrose syrup was observed among all bee groups throughout the 21 days. Furthermore, the nutritional responses of nurse bees—specifically, the expressions of major royal jelly protein 1 (mrjp1) in the head and vitellogenin (vg) in the abdomen—upon the provision of five single-supplemental diets were evaluated. We aimed to improve the usability of bee cages by elucidating the food consumption of bees and the relationship between nutritional gene expression and the ingestion of each single-protein diet.

Mechanistic Insight into Royal Protein Inhibiting the Gram-Positive Bacteria.

Royal jelly (RJ), a natural honeybee product, has a wide range of antibacterial activities. N-glycosylated major royal jelly protein 2 (N-MRJP2), purified from RJ, can inhibit the growth of Paenibacillus larvae (P. larvae, Gram-positive), a contagious etiological agent of the American foulbrood disease of honeybees. However, the inhibitory mechanism is largely unknown. Antibacterial assay and membrane proteome were conducted to investigate the inhibition capacity of RJ from different instar larvae and P. larvae treated by N-MRJP2, respectively. The similar antibacterial efficiency of RJ from different larval instar indicates that RJ is vital for the adaptive immune defense of small larvae. The killing of P. larvae by N-MRJP2 is achieved by disturbing the cell wall biosynthesis, increasing the permeability of cell membrane, hindering aerobic respiration, restraining cell division and inducing cell death. This demonstrates that RJ is critical for the passive immunity of immature larvae and N-MRJP2 can be used as natural antibiotic substance to resist P. larvae, even for other gram-positive bacteria. This constitutes solid evidence that RJ and N-MRJP2 have potentials as novel antibacterial agents.

Quantification of major royal jelly proteins using ultra performance liquid chromatography tandem triple quadrupole mass spectrometry and application in honey authenticity

Major royal jelly proteins (MRJPs) have been used as endogenous biomarkers for honey authentication. However, no study on quantification of MRJPs in honey authentication was reported. In order to develop a new honey authenticity method based on quantitative analysis of MRJPs in honey, trypsin-digested peptides from MRJPs were examined by mass spectrometry (MS). Three peptides, YNGVPSSLNVISK, TLQMIAGMK, and LTVAGESFTVK were selected as specific markers for MRJP1, MRJP 2 and MRJP 3(MRJP1∼3), respectively. An ultra performance liquid chromatography-triple quadrupole MS (UPLC-TQMS) method used for quantification of MRJPs in honey was developed and validated. A database on the MRJP1∼3 contents of honey established by quantification of 70 authentic samples showed a potential application in honey authenticity identification. The analysis results of 10 commercial honey samples by UPLC-TQMS with reference to the MRJP1∼3 database of authentic honey were compared and confirmed by widely used isotope ratio mass spectrometry, a AOAC Official Method. In conclusion, the results showed our method, a proteomic-based UPLC-TQMS method, has a high accuracy, sensitivity and specificity for honey authenticity.

Supplementation with an Inorganic Zinc Source in the Metalloproteomic Profile of Royal Jelly in Apis mellifera L.

This study aimed to evaluate the quality of royal jelly produced by honeybees Apis mellifera supplemented with different concentrations of inorganic zinc (zinc sulfate monohydrate-0, 25, 50, and 75 ppm). Two-dimensional electrophoresis for the fractionation of royal jelly proteins was performed, and the zinc level was quantified by the flame atomic absorption spectrometry (FAAS) technique. Proteins were identified by electrospray ionization mass spectrometry (ESI MS MS). Analysis of variance followed by the Tukey test (P < 0.05) was used. Supplementation with the mineral zinc positively affected the quantification of proteins for treatments 50 and 75 ppm. However, all treatments independent of zinc concentrations showed fewer protein spots when compared to the control. All zinc-containing proteins were classified as major royal jelly proteins (MRJPs). The exposure of nursing bees to the mineral zinc in its inorganic form reduced the expression of six different MRJPs involved in larval and glands development of nursing bees (MRJP1, MRJP2, MRJP3, MRJP5, and MRJP7), however promoted an increase in the expression of royal jelly proteins involved in defense systems (MRJP8 and MRJP9). The results demonstrate that vital proteins and metabolic processes are impaired in nursing bees exposed to the mineral zinc in its inorganic form in all doses used affecting nutrition and maintenance of colonies.

Comparative quantification and differentiation of bracatinga (Mimosa scabrella Bentham) honeydew honey proteins using targeted peptide markers identified by high-resolution mass spectrometry

Honey traceability is an important topic, especially for honeydew honeys, due to the increased incidence of adulteration. This study aimed to establish specific markers to quantify proteins in honey. A proteomics strategy to identify marker peptides from bracatinga honeydew honey was therefore developed. The proteomics approach was based on initial untargeted identification of honey proteins and peptides by LC-ESI-Triple-TOF-MS/MS, which identified the major royal jelly proteins (MRJP) presence. Afterwards, the peptides were selected by the in silico digestion. The marker peptides were quantified by the developed targeted LC-QqQ-MS/MS method, which provided good linearity and specificity, besides recoveries between 92 and 100% to quantify peptides from bracatinga honeydew honey. The uniqueness and high response in mass spectrometry were backed by further complementary protein analysis (SDS-PAGE). The selected marker peptides EALPHVPIFDR (MRJP 1), ILGANVK (MRJP 2), TFVTIER (MRJP 3), QNIDVVAR (MRJP 4), FINNDYNFNEVNFR (MRJP 5) and LLQPYPDWSWTK (MRJP 7), quantified by LC-QqQ-MS/MS, highlighted that the content of QNIDVVAR from MRJP 4 could be used to differentiate bracatinga honeydew honey from floral honeys (p < 0.05) as a potential marker for its authentication. Finally, principal components analysis highlighted the QNIDVVAR content as a good descriptor of the analyzed bracatinga honeydew honey samples.

The Carboxyl-Terminal Penta-Peptide Repeats of Major Royal Jelly Protein 3 Enhance Cell Proliferation.

Royal jelly (RJ) is known as an important functional foodstuff that promotes several health benefits and contains various bioactive substances, including major royal jelly proteins (MRJPs). Among the MRJPs, MRJP3 possesses both cell proliferation and wound healing effects. As the carboxyl domain of MRJP3 contains tandem penta-peptide repeat (TPR) sequences unique to MRJP3 among the MRJPs, we purified the TPRs as glutathione-S-transferase (GST)-fusion proteins and demonstrated their dose-dependent effects on THP-1 and Vero cell proliferation. The GST-TPR protein with 19 repeats (GST-TPR19) showed cell proliferative activity equivalent to MRJP3 and higher than GST-TPR6. GST-TPR19 also exhibited wound healing activity at a level similar to MRJP3. Digestion of GST-TPR19 with trypsin had no effect on its cell proliferative activity, suggesting that the main digested products; i.e., penta-peptides (Q-N-x-N-[K/R]), maintain the cell proliferative ability of MRJP3. In conclusion, the TPRs of MRJP3 are critical to the beneficial effect(s) of RJ.

Structure of native glycolipoprotein filaments in honeybee royal jelly

Royal jelly (RJ) is produced by honeybees (Apis mellifera) as nutrition during larval development. The high viscosity of RJ originates from high concentrations of long lipoprotein filaments that include the glycosylated major royal jelly protein 1 (MRJP1), the small protein apisimin and insect lipids. Using cryo-electron microscopy we reveal the architecture and the composition of RJ filaments, in which the MRJP1 forms the outer shell of the assembly, surrounding stacked apisimin tetramers harbouring tightly packed lipids in the centre. The structural data rationalize the pH-dependent disassembly of RJ filaments in the gut of the larvae.

Carbendazim exposure during the larval stage suppresses major royal jelly protein expression in nurse bees (Apis mellifera)

Studying the sublethal effects of agrochemical pesticides on nontarget honeybees (Apis mellifera) is important for agricultural development. Carbendazim is a widely used broad-spectrum fungicide that inhibits mitotic microtubule formation and cell division. However, the impact of carbendazim on bee health and development has not been fully elucidated. Here, using proteomics approaches, we assessed in vitro the changes in the expression of functional proteins in the head of newly emerged adults following treatment with field concentration of carbendazim during the larval stage. Treatment with carbendazim severely altered 266 protein expression patterns in the heads of adults and 218 of them showed downregulation after carbendazim exposure. Notably, major royal jelly proteins, a crucial multifunctional protein family with irreplaceable function in sustaining the development of colonies, were significantly suppressed in carbendazim-treated bees. This result was verified in both head and hypopharyngeal gland of nurse bees. Moreover, visual and olfactory loss, immune functions, muscular activity, social behavior, neural and brain development, protein synthesis and modification, and metabolism-related proteins were likely inhibited by carbendazim treatment. Together, these results suggest that carbendazim is an environmental risk factor that likely weakens bee colonies, partially due to reduced expression of major royal jelly proteins, which may be potential causes of colony collapse disorder.

The potential antiviral effect of major royal jelly protein2 and its isoform X1 against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Insight on their sialidase activity and molecular docking.

Severe acute respiratory syndrome-coronavirus (SARS-CoV)-2 is a newly emerging type of CoV. We evaluated the predicted anti-SARS-CoV-2 effect of major royal jelly protein (MRJP)2 and MRJP2 isoform X1, which recently showed high efficacy against other enveloped RNA-viruses (HCV and HIV). Some in-silico analyses have been performed to predict the impact of these proteins on viral entry, replication, and complications. These proteins have shown a high potency in sialic acid hydrolysis from the lung cells (WI-38) surface. Docking analysis showed that these proteins have a high binding affinity to viral receptor-binding sites in the receptor-binding domain, causing attachment prevention. Moreover, MRJPs can exert an inhibitory influence, via different mechanisms, for SARS-CoV-2 non-structural proteins (main and papain proteases, RNA replicase, RNA-dependent RNA polymerase, and methyltransferase). Also, they can bind to hemoglobin-binding sites on viral-nsps and prevent their hemoglobin attack. Thus, MRJP2 and MRJP2 X1 can be a promising therapy for SARS-CoV-2 infection.

Ethanol-soluble proteins from the royal jelly of Xinjiang black bees.

Royal jelly is a nutritious food that has beneficial effects to human health. However, the functional substances remain unclear. Herein, we fractioned the royal jelly proteins of Xinjing black bees according to the Osboren method. Two main proteins from the ethanol-soluble fraction were purified and identified. RJG-1 was determined as glucosylceramidase, and RJG-2 was major royal jelly protein 1 (MRJP1). RJG-1 showed potent cytotoxicity toward various mammalian cells, and caused quick disruption of cell membranes. With glucosylceramidase activity, RJG-1 may degrade the glucosylceramide of the cell membranes and disrupt the membrane structure, thereby resulting in cell necrosis. This study extends our knowledge about the composition and function of royal jelly, and is significant for the application of royal jelly.

Proteomic analysis of honey. Identification of unique peptide markers for authentication of NZ mānuka (Leptospermum scoparium) honey

Proteomics is an emerging tool in food authentication that has not been optimised for honey analysis. In this study, we present a qualitative proteomic analysis of New Zealand mānuka (Leptospermum scoparium) honey. A total of fifty bee-derived proteins were identified in the honey, the most predominant being major royal jelly proteins (MRJPs). We also demonstrate for the first time the presence of unique nectar-derived proteins in mānuka honey. A total of 17 mānuka plant proteins were identified, a-third of which were putative pathogenesis-related proteins. Two proteins involved in drought tolerance were also identified. Twelve candidate peptides were selected as potential authentication markers based on their uniqueness to mānuka honey. Nectar analyses confirmed the origin and specificity of these peptides to L. scoparium nectar, thus presenting peptide profiling as a viable and novel approach for mānuka honey authentication. Raw data are available via ProteomeXchange with identifier PXD021730.

Changes in Honey Bee Head Proteome in Response to Dietary 24-Methylenecholesterol.

Simple SummaryPhytosterols are important micronutrients that are essential for production of insect molting hormones and cellular membrane integrity. Past research has shown that the key phytosterol that honey bees need is 24-methylenecholesterol. This phytosterol improves honey bee longevity and sustains brood production. Hence, it is important to understand how 24-methylenecholesterol can shape honey bee physiology by altering protein profiles of vital honey bee tissues. Nurse bees secrete glandular secretions (brood food) using hypopharyngeal and mandibular glands in their head regions. Further, it has been shown that this sterol is selectively accumulated in nurse bee heads. Thus, it is imperative to examine the protein profiles of nurse bee heads, in response to dietary 24-methylenecholesterol manipulation. In this study, groups of newly emerged nurse bees were fed with varying concentrations of dietary 24-methylenecholesterol, while the control groups received no sterol. We found that dietary sterol manipulation altered the protein profiles in nurse bee heads, with important nutritional marker proteins being upregulated in high dietary sterol groups. The important proteins identified in this study may serve as vital markers of nutritional stress related to sterols in honey bees, paving the way for future research on bee nutrition.Phytosterols are important micronutrients that are precursors of important molting hormones and help maintain cellular membrane integrity in insects including bees. Previous research has shown that 24-methylenecholesterol is a key phytosterol that enhances honey bee longevity and improves nurse bee physiology. Nurse bees have the ability to selectively transfer this sterol to developing larvae through brood food. This study examines the physiological impacts of 24-methylenecholesterol on nurse bees, by analyzing the protein profiles of nurse bee heads upon dietary sterol manipulation. Dietary experimental groups consisting of newly emerged honey bees were provided with varying concentrations of 24-methylenecholesterol for three weeks. At the end of the study, honey bees were collected and proteomic analysis was performed on honey bee heads. A total of 1715 proteins were identified across experimental groups. The mean relative abundances of nutritional marker proteins (viz. major royal jelly proteins 1, 4, 5, 7) were higher in experimental groups supplemented with higher dietary sterol concentrations, when compared with the control dietary group. The mean relative abundances of important enzymatic proteins (aminopeptidase and calcium-transporting ATPase) were higher in control groups, whereas mean relative abundances of oxysterol-binding protein and fatty acid-binding protein were higher in higher dietary sterol groups.

The Neuroproteomic Basis of Enhanced Perception and Processing of Brood Signals That Trigger Increased Reproductive Investment in Honeybee (Apis mellifera) Workers

The neuronal basis of complex social behavior is still poorly understood. In honeybees, reproductive investment decisions are made at the colony-level. Queens develop from female-destined larvae that receive alloparental care from nurse bees in the form of ad-libitum royal jelly (RJ) secretions. Typically, the number of raised new queens is limited but genetic breeding of "royal jelly bees" (RJBs) for enhanced RJ production over decades has led to a dramatic increase of reproductive investment in queens. Here, we compare RJBs to unselected Italian bees (ITBs) to investigate how their cognitive processing of larval signals in the mushroom bodies (MBs) and antennal lobes (ALs) may contribute to their behavioral differences. A cross-fostering experiment confirms that the RJB syndrome is mainly due to a shift in nurse bee alloparental care behavior. Using olfactory conditioning of the proboscis extension reflex, we show that the RJB nurses spontaneously respond more often to larval odors compared with ITB nurses but their subsequent learning occurs at similar rates. These phenotypic findings are corroborated by our demonstration that the proteome of the brain, particularly of the ALs differs between RJBs and ITBs. Notably, in the ALs of RJB newly emerged bees and nurses compared with ITBs, processes of energy and nutrient metabolism, signal transduction are up-regulated, priming the ALs for receiving and processing the brood signals from the antennae. Moreover, highly abundant major royal jelly proteins and hexamerins in RJBs compared with ITBs during early life when the nervous system still develops suggest crucial new neurobiological roles for these well-characterized proteins. Altogether, our findings reveal that RJBs have evolved a strong olfactory response to larvae, enabled by numerous neurophysiological adaptations that increase the nurse bees' alloparental care behavior.

Colony-level pesticide exposure affects honey bee (Apis mellifera L.) royal jelly production and nutritional composition

Honey bees provision glandular secretions in the form of royal jelly as larval nourishment to developing queens. Exposure to chemicals and nutritional conditions can influence queen development and thus impact colony fitness. Previous research reports that royal jelly remains pesticide-free during colony-level exposure and that chemical residues are buffered by the nurse bees. However, the impacts of pesticides can also manifest in quality and quantity of royal jelly produced by nurse bees. Here, we tested how colony exposure to a multi-pesticide pollen treatment influences the amount of royal jelly provisioned per queen and the additional impacts on royal jelly nutritional quality. We observed differences in the metabolome, proteome, and phytosterol compositions of royal jelly synthesized by nurse bees from multi-pesticide exposed colonies, including significant reductions of key nutrients such as 24-methylenecholesterol, major royal jelly proteins, and 10-hydroxy-2-decenoic acid. Additionally, quantity of royal jelly provisioned per queen was lower in colonies exposed to pesticides, but this effect was colony-dependent. Pesticide treatment had a greater impact on royal jelly nutritional composition than the weight of royal jelly provisioned per queen cell. These novel findings highlight the indirect effects of pesticide exposure on queen developmental nutrition and allude to social consequences of nurse bee glandular degeneration.

Recuperación parcial de la proteína MRJP1 expresada en Pichia pastoris utilizando técnicas cromatográficas

Major Royal Jelly Protein 1 (MRJP1) is the main protein component of the bee-produced complex mixture royal jelly, which is the only nutrient source for queen bees promoting increased lifespan, body size and fertility. Recombinant production of MRJP1 represents an alternative to direct extraction from royal jelly. Production in Pichia pastoris results in high density biomass, with a supernatant containing high amount of impurities. Various methods have been applied to recover and/or purify MRJP1. Here, exploiting the physicochemical properties of MRJP1, reverse phase chromatography (RPC), size exclusion chromatography (SEC) and ion-exchange chromatography (IEX) were investigated as alternative methods to recover MRJP1 directly from supernatant. All techniques showed a 57-kDa band in SDS-PAGE analysis, corresponding to the size of recombinant MRJP1, with contaminants attributed to culture media. However, SEC coupled to IEX evidenced a single peak in the chromatogram corresponding to MRJP1 which suggest it may be a good protocol to recover recombinant MRJP1 from P. pastoris supernatant. This approach serves as a procedure to identify MRJP1 in fermentation culture of P. pastoris. This is the first report about characterization of IEX-based recovery of recombinant Apis mellifera MRJP1 produced in Pichia pastoris without the use of histidine tags.

Insecticides cause transcriptional alterations of endocrine related genes in the brain of honey bee foragers

Bees are exposed to endocrine active insecticides. Here we assessed expressional alteration of marker genes indicative of endocrine effects in the brain of honey bees. We exposed foragers to chlorpyrifos, cypermethrin and thiacloprid and assessed the expression of genes after exposure for 24 h, 48 h and 72 h. Chlorpyrifos caused the strongest expressional changes at 24 h characterized by induction of vitellogenin, major royal jelly protein (mrjp) 2 and 3, insulin-like peptide (ilp1), alpha-glucosidase (hbg3) and sima, and down-regulation of buffy. Cypermethrin caused minor induction of mrjp1, mrjp2, mmp1 and ilp1. The sima transcript showed down-regulation at 48 h and up-regulation at 72 h. Exposure to thiacloprid caused down-regulation of vitellogenin, mrjp1 and sima at 24 h, and hbg3 at 72 h, as well as induction of ilp1 at 48 h. The buffy transcript was down-regulated at 24 h and up-regulated at 48 h. Despite compound-specific expression patterns, each insecticide altered the expression of some of the suggested endocrine system related genes. Our study suggests that expressional changes of genes prominently expressed in nurse or forager bees, including down-regulation of buffy and mrjps and up-regulation of hbg3 and ilp1 may serve as indicators for endocrine activity of insecticides in foragers.

Major royal jelly proteins accelerate onset of puberty and promote ovarian follicular development in immature female mice

The major royal jelly proteins (MRJPs) are the central constituents responsible for the specific activities of royal jelly. Here MRJPs via oral administration daily for 45 consecutive days were evaluated the effects on the reproductive parameters in immature female mice (FM). Neonatal FM were divided into four groups fed MRJPs with doses of 0, 125, 250 and 500mg/kg/body weight (M125, M250 and M500). The results in M125, M250 and M500 showed that the times of estrus were accelerated by 10.7%, 15.5% and 10.7%, the secondary follicles number were increased by 50.7%, 78.8% and 38.6%, the Graafian follicles were increased by 600.0% and 774.0% and 150.0%, respectively. M500 induced multi-oocyte follicles. The serum estradiol levels of the three groups were increased by 47.1%, 64.9% and 31.1%, the action of MRJPs raising hormone secretion level is mainly via upregulating expression of ERβ gene. Antioxidant parameters of ovarian tissue showed that the malondialdehyde levels in M125 and M250 were decreased, the superoxide dismutase activities and glutathione peroxidase activities in M125 and M250 were increased. In conclusion, MRJPs may accelerate onset of puberty and promote follicular development in FM. Our findings would facilitate better understanding of the benefit effect of MRJPs as the key ingredient in royal jelly on promoting fertility performance.

Purification and characteristics of individual major royal jelly protein 1–3

Major royal jelly proteins (MRJPs) are deemed to the most characteristic and abundant constituents in royal jelly. The present paper establishes a sequential process to purify the natural MRJP 1–3 by three different chromatographic procedures. Five individual proteins were obtained, including MRJP 1 oligomer 1, MRJP 1 oligomer 2, MRJP 1 monomer, MRJP 2 and MRJP 3s (contain three MRJP 3 variants) with a purity of 89.88%, 67.79%, 99.92%, 99.41%, and 99.97%, respectively. This is the first report that MRJP 1 mainly possesses three oligomers: a 228 kDa MRJP 1 oligomer 1, a 408 kDa MRJP 1 oligomer 2 and a 639 kDa MRJP 1 oligomer 3. We infer that MRJP 1 oligomer 3 (639 kDa) may be assembled by MRJP 1 oligomer 1 (228 kDa) and MRJP 1 oligomer 2 (408 kDa). The secondary structure of the purified MRJP 1–3 consisted predominantly of β-sheets and random coil in the native conformation. Our results will contribute to the physiological and physicochemical function of the purified individual MRJPs.