Expression, Purification And Biological Activities Of PP-rhBMP-4m

E. coli expression system, which is divided into fusion and non-fusion expression, is frequently used for genetic engineering way of protein preparation. To fuse the gene sequence of coding target protein with the sequence of the expression vector which can highly express the known protein or polypeptide is called fusion expression system. There are some advantages of this system. First, it is easy to achieve high level expression of target protein. Second, the hydrophilicity or the characteristic of molecular chaperones of the fusion protein or polypeptide can make the dissolving or regeneration of the target protein easily. Third, for the various tags of the fusion expression, the target protein can be easily purified and identified. Therefore, fusion expression system has become the popular way for the study of protein function and genetic engineered protein drugs. Plenty of sale protein fusion expression vectors have been created and applied widespreadlydd. However, there are some common disadvantages of the most fusion expression vectors used today. For example, the tag protein of target protein can not be removal easily; the tag protein in vivo can cause unnecessary immunoreactions; the activity and the normal behavior of the target protein will be influenced etc. Enzymology methods are frequently used to remove the tag protein, but this way is costly, unefficiency and with low recovery rate of target protein. Chemical cleavage ways, such as cyanogen bromide are poison and seldom used.Bone morphogenetic protein 4 (BMP-4) is a member of the BMPs family. Human BMP-4 mature peptide (hBMP-4m) is composed of 116 amino acids. BMP-4 has many physiologic functions including bone generation and hematopoietic tissue induction. BMP-4 is a growth factor with long time effects on promoting the growing of all three-line haemocytes and its roles are more permanent than other BMPs. The preparation of BMP-4 from eukaryotic expression system can obtain BMP-4 with good activity. But for its low yield and high cost, it can only be used in labs and is restricted for clinical application. E. coli system with high yield and simple technology for production and purification, it can still be considered to be a choice. We have cloned the cDNA coding sequence of hBMP-4m and cloned it into a expression vector named pDH2. It was expressed and purified by non-fusion system in E. coli (which has been obtained the national invention patent), but the purified procedures were complex and the recovery and the yields rate were quite low.We tried to find a expression system by which the recombinant protein was not only over expressed but also purified easily, and the recombinant protein should have the similar biological function with the native protein. We constructed successfully the E.coli formic acid acidolysis fusion expression vector, using hBMP-4m as a target. This system also can be used for other protein expression and purification.1. The construction of the prokaryotic formic acid acidolysis fusion expression vectorAim: To construct a formic acid acidolysis fusion expression vector by which the target protein could over expressed. Methods: The different synonymous codons of Asp-Pro-Pro which contained the formic acid acidolysis site we designed were added to 5'-teminal of hBMP-4m colding sequence by PCR. The sequences were cloned into the pRSET-B fusion expression vectors, then the plasmids were transformed into E.coli BL21 and induced to express. The expressed fusion protein was analyzed by SDS-PAGE. Results: The nine fusion expression vectors with different codons of Asp-Pro-Pro were constructed and named pRSET-DPP-rhBMP4m 1~9 respectively. The fusion protein could be expressed efficiently only when the codons of the two Pro were both CCG. And the percentage of the fusion protein expressed reached 38.4% of the total bacterial proteins. Conclusion: The yields of the fusion protein can be influenced significantly by the changing of the sequences in the middle region of the peptide chain.2. The application of the formic acid acidolysis fusion expression systemAim: To express the PP-rhBMP-4m in large scale using the formic acid acidolysis fusion expression system we constructed , and purify the target protein. Methods: The PP-rhBMP-4m engineering strains (BL21/pRSET-DPP-rhBMP-4m) were growth and induced to express in a 15 L automatic control fermantation device. The resulting inclusive bodies were washed and dissolved in 8M urea denatured buffer. The fusion protein was purified directly with nickel ion affinity column, and then removed 6×His-D tag peptide protein using formic acid acidolysis. The target protein was analyzed by SDS-PAGE. And the yield of the target protein was calculated. Results: The percentage of the fusion protein after fermented and washed accounted for 57% of the total proteins. After affinity chromatograph, the purity of the target protein was 98%. The yield of the target protein was 69.8%. The recovery of the target protein after acidolysis was 93%. Conclusion: The pRSET-DPP-rhBMP-4m we constructed was an efficient and convenient purifying fusion expression system.3. The detection of the PP-rhBMP-4m cytoactiveAim: To detect the difference on biological activity between the target protein PP-rhBMP-4m which was involved two Pro at the amino terminus and the non-fusion protein rhBMP-4m. Method: The mesenchymal stem cells (MSCs) were induced by PP-rhBMP-4m and rhBMP-4m respectively, and then the cell proliferation with MTT method was observed and the change of the amount of ALP in MSCs was detected. The C2C12K5 and NIH3T3K2 cells (which have been obtained the national invention patent No. ZL 01 1 31813.9) which contained the luciferase report gene were stimulated by PP-rhBMP-4m and rhBMP-4m respectively, and the changes of the luciferase activities were measured. Results: Both the two kinds of protein at the same concentration promoted MSCs proliferation and ALP in MSCs increased with the similar feature. There was no significant difference between the two groups. The results also demonstrated the luciferase activities were increased in the same appearance both in C2C12K5 and NIH3T3K2 when they stimulated. Conclusion: PP-rhBMP-4m and rhBMP-4m have no differences in cytoactivities.We have designed the tri-peptide sequence, Asp-Pro-Pro, according to the phenomena that Asp-Pro offers a site for formic hydrolysis and the prolyl peptidases which can remove the N-terminal Pro-Pro- of protein in mammalian ubiquitous. We used the tri-peptide to linkage the tag protein and the target protein and got the structure of"tag protein-Asp-Pro-Pro-target protein". After such peptide expressed in high level, we can purify the target protein easily by affinity chromatography firstly. Then, the protein can be removed the tag peptide by formic hydrolysis with high efficiency. When the resulting target protein carried Pro-Pro at the amino terminus was put into animal bodies, Pro-Pro- will be removed by prolyl peptidase and the target protein will show its biological effects. Moreover, this formic hydrolysis fusion expression system can be used for the expression and purification of many kinds of proteins especially for the expression of small peptide. By this structure of Asp-Pro-Pro we can create a small peptide concatemer, which can overcome the troubles on the expression and detection of small peptide. And we can get the monomers of small peptide when we use this structure of Asp-Pro-Pro to purify the target protein. This construction is a good method for the prokaryotic expression and high efficiency purification of small peptide.