|SHEN, LI-RONG - Zhejiang University|
|ZHANG, WEI-GUNGAN - Zhejiang University|
|JIN, FENG - Zhejiang University|
|ZHANG, LI-WEN - Zhejiang University|
|CHEN, ZHEN-XIAN - Zhejiang University|
|LIU, LIANG - Delaware State University|
|Lai, Chao Qiang|
|LI, DUO - Zhejiang University|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2010
Publication Date: 2/24/2010
Citation: Shen, L., Zhang, W., Jin, F., Zhang, L., Chen, Z., Liu, L., Parnell, L.D., Lai, C., Li, D. 2010. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line. Journal of Agricultural and Food Chemistry. 58(4):2266-2273.
Interpretive Summary: That a honeybee queen lives over ten times longer than worker bees has been attributed to her longer feeding time of royal jelly (RJ) during the larval development. RJ has a wide range of potential biological functions in human health, including blood vessel constriction, lowering blood pressure, and anti-tumor, anti-inflammatory, disinfectant, and anti-fatigue activities. Moreover, RJ can increase the average lifespan of mice. Due to its richness of a large number of bioactive substances, RJ has been used widely as a popular supplement for health promotion and cosmetic applications. In order to identify the potent components of RJ, its main protein constituent, called main royal jelly protein 1 (MRJP1), has been identified from the Western honeybee and its DNA copied for further study. In the present study, we have cloned the MRJP1 gene (AccMRJP1) from the Chinese honeybee, and expressed it in a yeast strain, called Pichia pastori. Expression in this yeast was verified by standard protein analysis tools and showed furthermore that the cloned AccMRJP1 is indeed the same protein of the Western honeybee MRJP1. The molecular weight of the Chinese honeybee Acc MRJP1 protein decreased from 57 kDa to 48 kDa after enzymatic treatment that breaks sugars from proteins, indicating that the AccMRJP1 protein was indeed decorated with specific sugar-like molecules. Our results also showed that AccMRJP1 significantly stimulated the growth of Tn-5B-4 insect cells in a manner similar to the expensive combination of AmSPRJ (a mixture of other, lesser royal jelly proteins) and fetal bovine serum that affected both cell shape and adhesion to the dish in which these cells were grown. Together, these results provide a foundation for experiments that can be conducted to fully characterize the mechanisms by which AccMRJP1 exerts its biological activities.
Technical Abstract: Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in molecular weight to the glycosylated AmMRJP1 from the Western honeybee (A. mellifera). Western blots probed with the anti-AccMRJP1 antibody demonstrated that recombinant AccMRJP1 and soluble protein of the Western honeybee RJ (AmSPRJ) contained immunoreactive MRJP1. The 57 kDa protein in AmSPRJ contained an N-terminal amino sequence of N-I-L-R-G-E which is identical to that previously characterized in AmMRJP1. The molecular weight of recombinant AccMRJP1 was decreased from 57 kDa to 48 kDa after deglycosylation, indicating that AccMRJP1 was glycosylated. The recombinant AccMRJP1 significantly stimulated Tn-5B-4 cell growth similar to AmSPRJ and fetal bovine serum, and affect cell shape and adhesion to the substrate.