Heterogeneous Expression And Functional Characterization Of Oxysterol-binding Protein From Aspergillus Oryzae

Aspergillus oryzae is a GRAS-level safe strain that brews traditional soy sauce and rice wine,and has a history of more than one thousand years of safe application.Although the genome of A.oryzae was sequenced in 2005,there were many functional important genes needed for further exploretion.Oxysterol-binding proteins are a class of conserved proteins that bind to oxidized sterols and present in eukaryotic organisms.It participates in cell lipid metabolism,vesicular transport and signal transmission and has other important functions.More importantly,a large number of studies have shown that these proteins have the ability to bind cholesterol and phytosterols,which is the key point why we choose to give further insight into the Oxysterol-binding protein(osbp).In this study,we chose the Oxysterol-binding protein derived from A.oryzae 3.042 as the research object.The results were as follows:1)The bioinformatics analysis showed that Oxysterol-binding protein of Aspergillus oryzae(AoOSBP)was a typical conserved protein.Also the ligand binding domain of one of its functional domains was quite conserved,which has a fingerprint sequence EQVSHHPP and could be used to search Oxysterol-binding protein homologue.AoOSBP shares 99% similarity to its homologue Oxysterol-binding protein gene of Aspergillus flavus.2)The expression patterns of Oxysterol-binding protein in Aspergillus oryzae were determined from the gene level to the expression level by using qRT-PCR,Western blot and immunofluorescence.The results of qRT-PCR showed that the gene expression level of AoOSBP did not have obvious change at the time of 24 h,36h,48 h,60h and 72 h,but the gene expression level of AoOSBP at 72 h was a little higher than that of 24 h.Western blot analysis showed that the expression of AoOSBP increased slightly at 48 h and 60 h.But the overall increase of protein expression is not obvious.Immunofluorescence experiments can be used to show that the Oxysterol-binding protein moves at different growth points.At 24 h,AoOSBP was mainly concentrated in the nucleus,and then to 36 h and 48 h,began to spore production,we can see the distribution of the protein to the whole mycelium and the new spores.At 60 h and 72 h,the spores were already mature and observed that the protein specifically detected in mycelium was highly expressed.Considering the above results,there was high expression of Oxysterol-binding protein in A.oryzae at different growth periods.3)The expression vectors pET30a-osbp and pMAL-c4X-osbp-ord were constructed to express AoOSBP and the OSBP-related ligand-binding domain of Oxysterol-binding protein of A.oryzae(AoOSBP-ORD),respectively.Under the same induction conditions,the expression of AoOSBP by pET30a-osbp recombined vector did not increase significantly after induction,and was demonstrated by the Western blot.However,the expression of AoOSBP-ORD expressed by pMAL-c4X-osbp-ord vector increased significantly after induction,and AoOSBP-ORD was purified by an amylose resin chromatography column.4)The function of purified AoOSBP and AoOSBP-ORD expressed in prokaryotic cells were analyzed by Microscale thermophoresis binding assay.The AoOSBP binding experiments show that the binding ability of Ergosterol(Kd = 74.338 nM),25-hydroxycholesterol(Kd = 29.899 nM),7-keto Cholesterol(Kd = 7.772 nM),Soybean Sterol(Kd = 24.717 nM),Stigmasterol(Kd = 32.469 nM),vegetable oil sterol(Kd = 20.328 uM)and ?-sitosterol(Kd = 27.521 uM),respectively.The smaller the Kd value,the stronger the binding ability of the protein to the substrate,and the combination of their Kd values shows that the protein in addition to vegetable oil and ?-sitosterol substrate has a strong ability to bind.However,when we used AoOSBP-ORD to validate the above function of full-length protein,AoOSBP-ORD has no binding ability to vegetable sterol and ?-sitosterol and has a strong ability to bind several other sterol substrates.Thus,AoOSBP can act as a carrier for sterol transport.Oxidizing sterols and phytosterols have a role in reducing cholesterol,and AoOSBP is also of important application value in the development of cholesterol-lowering foods and drugs.