Study On The Purification And Anti-tumor Activity Of Protein Produced By The Endophyte DNN7 Isolated From Laminaria Japonica Aresch

The marine microorganisms could produce the metabolites with anti-tumor activity, while there are few of report about metabolites produced by the endophyte isolated from Laminaria japonica Aresch. So it is necessary to research on biology activity of metabolites produced by the endophyte isolated from Laminaria japonica Aresch. In this paper, the crude protein produced by endophyte DNN7 isolated from Laminaria japonica Aresch was researched. The DEAE 52 anion-exchange chromatography combined with Sephadex G-100 molecular sieve chromatography was used to separate protein of DNN7. The protein concentration of separated components was determined. The separated components were also analysed by SDS-PAGE, MALDI-TOF/TOF-MS, and the anti-tumor activity of separated components were also invesgeted. The main results obtained are as follows:1. P1, P2, P3 and P4 were obtained after purication of DNN7 crude protein by DEAE 52 anion-exchange chromatography. P3 showed the highest antibacterial activity. SDS-PAGE revealed that there were only two bands in P1, and the bands of the other three separated components were more complex. The results of MALDI-TOF/TOF-MS indicated that the second band of P1, the first band of P2 and the second band of P3 which had been successfully matched in NCBI database, were identified as Subtilisin E, Tail tape measure protein gp18 and catalase resepectivly.2. P3-1, P3-2 and P3-3 were obtained after purication of P3 by Sephadex G-100 molecular sieve chromatography. The P3-3 showed better antibacterial activity. SDS-PAGE demonstrated there were 4 bands in P3-1 and 3 bands in P3-2, while single protein band in P3-3. The molecular weight of P3-3 was identificated by MALDI-TOF/TOF-MS. Research result showed that maybe there were double charge in P3-3. So the molecular weight of P3-3 is 8826.8 Da. The band of P3-3 failed to matcheing with known protein in the NCBI database, then it may be a kind of new protein.3. Results of MTT assay indicated that the inhibitory effect of P3 on human breast cancer cells MDA-MB-231 was firstly increased and then decreased during 10~300 μg/m L, and the highest inhibition ratio reached to 50.95% at the concentration of 200 μg/m L. P3-1, P3-2 and P3-3 also showed anti-tumor activity against human breast cancer cells MDA-MB-231, the inhibition ratio was 46.24%, 34.28% and 54.40% respectively. In addition, P3-3 could also inhibite human cervical cancer cells RL95 and hepatoma carcinoma cells HCC97 H, and the highest inhibition ratio is 25.36% and 32.65% respectively.