Structural Study Of The Royal Jelly Proteins MRJP1and MRJP2

MRJPs (Apis mellifera L.) are the important functional family proteins in royal jelly. Besidesserving as nutrient to queens and larvae, MRJPs also regulate the caste differentiation of bees and havestrong antimicrobial activity and other unknown functions. Therefore, many research groups carried outextensive research on MRJPs function. The function of biomacromolecule mainly depends on its spacestructure, but the structural information of MRJPs is rare, especially the research about protein X-raycrystallography and small Angle X ray scattering (SAXS) has not been reported yet. This paper focuseson the preliminary structural study of MRJP1and MRJP2protein, the main results are:First, The prokaryotic expression system and purification system of MRJP1was constructed, mrjp1gene was successfully inserted into expression vector pET-19b, the expression conditions andpurification system were optimized and the high purity MRJP1protein was obtained. At the same time,affinity chromatography, molecular sieve chromatography and ion exchange chromatography were usedto purify and obtain three kinds of single component. Two dimensional gel electrophoresis techniquesand MS-MS methods were used to identify them as MRJP1oligomer, MRJP1monomer and MRJP2,respectively. In addition, sieve chromatography, sedimentation equilibrium and SAXS were used toidentify MRJP1oligomer as MRJP1tetramer.Next, crystallization condition of five kinds of protein (recombinant MRJP1, MRJP1tetramer,MRJP1monomer, mixture of MRJP2and MRJP1monomer, MRJP2protein) were screened with sittingdrop vapor diffusion method. Three kinds of proteins (recombinant MRJP1, MRJP1monomer andMRJP2) were not found any protein crystals in384kinds of conditions. MRJP1tetramer crystal wasobtained in1.0mol/L Ammonium Phosphate,0.1mol/L Tris HCl pH8.5, then optimized with androd-shaped crystals were obtained which met the requirements of X-ray diffraction. The small needleand clusters crystals of mixture of MRJP2and MRJP1monomer were obtained in1.4mol/L SodiumAcetate,0.1mol/L Na Cacodylate pH6.5and then optimized with hanging drop method. Because of thelow resolutions with X-ray diffraction, none of the crystals data could be used for structure analysis.Again, secondary structures of three kinds of proteins (MRJP1tetramer, MRJP1monomer andMRJP2protein) were tested by synchrotron radiation circular dichroism (SRCD) technology. The resultshowed that the temperature has effect on the secondary structures of MRJP1tetramer, MRJP1monomer and MRJP2. The Tm value testing experiment showed that the denaturation temperature ofMRJP1tetramer was55℃, MRJP1monomer was42℃, and MRJP2was45℃. Furthermore, differentpH value, ion type and concentration also influenced the secondary structure of MRJP2.Finally, the3-D dot matrix structures was solved by using small angle X-ray scattering (SAXS)technology, MRJP1tetramer was a round pie, MRJP1monomer was a long rod and MRJP2was ahollow sphere. The error was small after the structure was verified, and the credibility was high.The results of this paper extended our knowledge about the structures of MRJPs and providedimportant foundation to discuss the function mechanism of royal jelly proteins.