Prokaryotic Expression Of MRJP2 From Apis Cerana Cerana

Honeybee was regarded as one of the members of insect families and a model organism for studying insect social behaviors. It was always honored as a research hot either in foreign and domestic. The Chinese honeybee Apis cerana cerana, one of the important subspecies of Asian honeybee, was considered as a precious source of honeybee in China and was adapted to the geographic and climate conditions of China. It had irreplaceable resources and research values.Royal Jelly (RJ) was secreted from the hypopharyngeal and mandibular glands of old nurse bees and newly emerged bee larvae were fed RJ for 3 days. The health care and biological functions of RJ had been reported and researches on its components and functions were always very important. Its functions were mainly generated by proteins presented in RJ. Royal Jelly proteins were comprised of water-soluble proteins and non-water-soluble proteins. Water-soluble proteins, also named Major Royal Jelly Proteins (MRJPs), represented 46%～89% of the total proteins in RJ. At present, study on RJ focused on MRJP families in molecular level.A full-length MRJP2 gene was screened from a cDNA library constructed from eight-day-old worker heads of Apis cerana cerana. The AccMRJP2 cDNA encompassed an Open Reading Frame (ORF) of 1407 bp encoding 468 amino acids with a predicted molecular mass of 53.06 kDa and a theoretical pI of 8.102. In this study, the entire ORF (1407 bp) was amplified from the AccMRJP2 cDNA by PCR and inserted into a prokaryotic expression vector pET-28a(+) to construct recombinant plasmid pET-28a(+)-AccMRJP2. Then the recombinant was transformed into Escherichia coli BL21(DE3) and induced by IPTG while the identification results of PCR, digestion with EcoR I/Xho I and sequence analysis showed validity. But SDS-PAGE results showed no specific bands even though several repeated experiments were performed. Then sub-cloning was considered. After removed the signal peptide sequence existed in AccMRJP2, the entire coding region (1356 bp) was divided into three parts with 420 bp, 486 bp, and 498 bp lengths respectively and amplified by PCR according to the primers designed. Each recombinant plasmid pET-28a(+)-AccMRJP2 was then constructed respectively and identified by PCR, digestion with Nde I/Xho I and sequence analysis. After the identification results showed that each fragment was inserted correctly, the fusion proteins His-420, His-486, His-498 and His-1356 were all expressed in E.coli BL21(DE3) and induced by IPTG. The molecular weight of each fusion protein was 19.7 kDa, 21.5 kDa, 22.4 kDa and 54.8 kDa (His tag was weight of 3.56 kDa) respectively. SDS-PAGE showed that His-420, His-486 and His-498 all had specific bands and expressed as inclusion bodies. However, His-1356 could not be expressed in E.coli. To further explore the optimal expression conditions for each protein, we optimized the expression conditions and designed 9 groups in different inducing time, inducing temperature and IPTG concentration. SDS-PAGE showed that the temperature rise from 30°C to 37°C could improve His-420 fusion protein expression, but had no effect on His-486 and His-498. Changes in the concentration of IPTG had no effect on each protein. This step also further confirmed they were all expressed as inclusion bodies. Then His-420, His-486 and His-498 could be purified by Ni-NTA affinity chromatography as they were all fused with His-tag. By affinity chromatography, high-purity proteins could be obtained. Then they were characterized by LC-MS mass-spectrum after being further purified by RP FPLC. The mass-spectrum results confirmed that His-420, His-486 and His-498 were all the products of AccMRJP2 and expressed in the right forms. In addition, a small peptide from the trypsin products of His-420, His-486 and His-498 was similar to the dipeptide Ile-Phe (IF, MW=278 Da) found in the gastrointestinal enzyme production of intact RJ by Matsui et al. This result suggested that the trypsin products from rAccMRJP2 were probably one of the sources of ACE inhibitory peptides.