Study On Prokaryotic Expression And Activity Of Protein MAP30 From Momordica Charantia

Momordica charantia was first recorded as a Chinese traditional medicine in the ancient book called the Materia Medica of south Yunnan,which was compiled by Lan Mao in the Ming Dynasty.It is a special plant that belongs to the food and medicine simultaneously,and it can remove pathogenic heat,alleviate fatigue,relax the mind and relieve visual fatigue.With the development of the modern extraction and separation technology,many kinds of components including saponins,polysaccharides,proteins and terpenoids with pharmacological activity have been extracted from it.Among them,a kind of isolated ribosome-inactivating protein called Momordica anti-HIV protein of 30 KD has become an attractive specific object of study for its broad spectrum of bacteriostasis,antiviral activity and antitumor activity,as well as its minimal cytotoxicity or cytostatic effect on uninfected target cells and other normal cells.However,it is not only cumbersome to extract MAP30 by traditional extraction and separation method,but also inefficient in the process of extraction,which cannot meet the needs of later research.So this study has combined traditional Chinese medicine and modern molecular technology to achieve the heterologous production of MAP30 using genetic engineering,to improve the yield of MAP30.MAP30 from heterologous expression was used for bacteriostasis experiment on some bacteria,the main results are as follows:(1)The bioinformatics analysis of MAP30:The molecular weight of MAP30 was about 32.02 kDa.Its number of amino acid residues was 286,and its theoretical isoelectric point was 9.08.MAP30 was a kind of stable protein according to prediction,whose instability index and estimated half-life of mammalian reticulocytes in vitro were 28.33 and 30 h,respectively.The prediction also showed that the signal peptide of MAP30 was MVKCLLLSFLIIAIFIGVPTAKG,which contained 23 amino acid residues.(2)TA cloning of MAP30:A pair of specific primers were firstly designed according to the MAP30 coding sequence.The gene encoding MAP30 was amplified by PCR,and the size of fragment was consistent with the expected size of about 900 bp.The fragment was recovered and cloned into pMD18-T cloning vector,and then transformed into Escherichia coli DH5?.After screening step by step,the cloned gene was finally sequenced and was aligned by Blast.Sequence comparison showed that the indentity of the reported sequence in GenBank and the cloned sequence was as high as 99.77%,which indicated that the MAP30 gene was successfully cloned.(3)Construction and induced expression of MAP30 prokaryotic expression engineering bacteria:pET28a,pET32 a and pMD18-T-MAP30,were digested respectively by Eco R I and Sal I,then MAP30 gene was ligated to expression vectors pET28 a and pET32 a separately.The recombinant expression plasmids pET28a-MAP30 and pET32a-MAP30 were respectively transformed into Escherichia coli BL21(DE)and Rosetta(DE3)pLysS.Finally two strains of recombinant prokaryotic engineering cells named pET32a-MAP30/Rosetta and pET28a-MAP30/BL21 were successfully screened.The electrophoresis of induced pET28a-MAP30/BL21 showed that no target protein band was consistent with the size of theoretical value by SDS-PAGE,indicating that the strain failed to express MAP30.The results of SDS-PAGE and Western blot of induced pET32a-MAP30/Rosetta could both reveal the target protein bands in line with the size of with the theoretical value,which manifested the strain could successfully express MAP30.The supernatant was collected after extending the fermentation scale of pET32a-MAP30/Rosetta,then the purified MAP30 solution was obtained by Ni-NTA affinity chromatography,and the final protein sample was obtained by desalting and freeze-drying.The fermentation yield of is 242 mg/L.(4)Study on the antimicrobial activity of MAP30:The antibacterial spectrum of MAP30 fusion protein by Oxford cup method included gram positive bacteria Staphylococcus aureus and Bacillus subtilis,gram negative bacteria Escherichia coli,and Saccharomyces cerevisiae.The minimum inhibitory concentration of fusion protein MAP30 on Staphylococcus aureus,Escherichia coli,Saccharomyces cerevisiae and Bacillus subtilis were 0.35 mg/mL,0.65 mg/mL,0.55 mg/mL and 0.75 mg/mL,respectively.Growth inhibition test showed fusion protein MAP30 had a certain inhibition on the normal growth of Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Saccharomyces cerevisiae.In summary,this study constructed a prokaryotic expression engineering strain that can successfully express MAP30 fusion protein,and researched the activity of the fusion protein systematically,which could lay the material foundation for heterologous production of MAP30 and the activity study of this protein.