Function Analysis Of BL0033 And BL0034 In Fructose ABC Transporter System Of Bifidobacterium Longum

The gastrointestinal tracts (GIT) of humans and other living organisms are colonized by a large, active and complex community of microbes, collectively termed intestinal microbiota. Bifidobacterium is considered to play an important role in the balance of normal intestinal flora. In addition, the contribution of Bifidobacterium to the reinforced host immune functions and improved resistance to cancer has been reported.At present, the bottleneck technology of industrialization production of fermenting the live fungus dairy products in vivo is that the live fungus was difficult to survive in a competitive environment of the human gastrointestinal tract.It is also the key question that must be solved on international development of Bifidobacterium industry. The nutrition of Bifidobacterium in vivo is mainly fructose-containing polymers. Thus, the compatibility of Bifidobacterium in intestinal tract will be revealed by its fructose transportation and metabolism mechanism. This study has certain theoretical significance and potential application values.In this study, several fructose transportation related proteins which were induced by fructose were obtained by using comprehensive proteomics and bioinformatics analysis.And genes of transportation proteins were cloned and expressed in E.coli BL21, substrate binded specificity were analyzed respectively in vitro. Interaction between fructose transportation system proteins were studied by GST-pull down method. The data were revealed as below:(1) Full cell proteins in Bifidobacterium longum NCC2705 were separated by two-dimensional gel electrophoresis (2-DE) with using pH (4-7) IPG strips and the spots distribution on general was understanded. A remarkable difference is observed that BL0033, probable solute binding protein of ABC transporter system possibly for sugars showed higher levels of expression in cells grown on fructose than on other sugars.(2) Expression vector pGEX-4T-1-bl0033 was constructed and massive fusion protein GST-BL0033 was expressed by E.coli BL21 and then purified by using glutathione-Sepharose the 4B resin followed by concentration and quantitation purified GST-BL0033 showed specific coherence of D-fructose.(3) Similarly, expression vector pGEX-4T-1-bl0034, massive fusion protein GST-BL0034 was expressed by E.coli BL21 and then GST-BL0034 was immobilized on glutathione-Sepharose 4B. After being incubated with ATP, it was found that BL0034 could bind ATP molecules.(4) Besides, both the genes of bl0033 and bl0034 in B. longum NCC2705 were successfully cloned into vector pET32a. Both two tagged forms of BL0033 and BL0034 were largely expressed in E.coli BL21 and then purified. Interactions between two proteins were analysised by GST-pull down method. Excitingly, obvious interaction was detected.All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific ABC transport system, in which BL0033 and BL0034 might play an important role.