Experimental Study Of A Novel Fusion Protein With Osteoinductive Activity

Therapy of bone defect is an important issue of basic research and clinical therapy. The development of bone tissue engineering brings a new method to the therapy of bone defect。The content of bone tissue engineering includes three elements: seed cells, scaffold and growth factors. Growth factors can promote seed cells proliferation and induce them to differentiate into cartilage cells or bone cells. Bone morphogenetic proteins (BMPs) have osteoinductive activity that can induce several kinds of seed cells to differentiate to osteoblasts. BMPs have been widely used in the areas of experimental research and clinical application. BMPs can be produced using eukaryotic expression system and prokaryotic expression system. The production prepared by the eukaryotic expression system has more effectively osteoinductive activity, but its clinical application is limited by the high cost. The product of the prokaryotic expression system is inclusion body which has no activity. After being renaturated, the BMPs molecules will be refolded and form dimer and recover their bioactivity. Because of the complicated renaturation, the efficiency of renaturation is fairly low and the activity is not stable. Although the activity has been elevated significantly using special renaturation method, the production cost price rised notablely. In clinical application, BMPs will not have osteoinductive activity until the accumulation of BMPs reaches some extent in local area. The expense will exceed the ability that patients can afford, so their clinical application is limited. In order to find a new cheaper protein having stable and high osteoinductive activity, many researches have been done.LMP1, an intracellular protein, has been discovered having osteoinductive activity recently. In some researches, its osteoinductive activity even exceeded the osteoinductive activity of BMPs. LMP1 has two splice variants. Through the analysis of the primary structure of the three splice variants, it can be found that the fragment of LMP1 (from the first amino acid to the 133th amino acid) is necessary for LMP1's osteoinductive activity.In this research, LMP1(1-133AA)is fused with protein transduction domain and the fusion protein is expressed by prokaryotic expression system. We postulate that the fusion protein can be transducted into cells and during the process, the fusion protein will be renaturated and recover its bioactivity. If the hypothesis proved to be truth, the fusion protein would be a novel growth factor with osteoinductive activity.Section 1: Construction of eukaryotic expression vector of LMP1 and the expression of LMP1 in HEK293 cellsIn this section, we constructed eukaryotic expression vector of LMP1 and observed the expression and localization of LMP1 in HEK293 cells. After the total RNA was extracted from the MG63 cells by Trizol method, LMP1 cDNA is acquired by reverse transcription-polymerase chain reaction (RT-PCR) using the primers designed and synthesized on the basis of the gene sequence of LMP1. The LMP1 cDNA is cloned into eukaryotic expression vector pEGFP-N3, and then we obtained the recombinant vector pEGFP-N3-LMP1. HEK293 cells were transfected with the vector pEGFP-N3-LMP1. It was found that the green flourescence only existed in cytoplasm in HEK293 cells observed by fluorescence microscope. So we confirmed that LMP1 is an intracellular protein which only exists in cytoplasm. LMP1 should interact with another protein in the cytoplasm and evoke the signal transduction pathway to promote bone formation.Section 2: Construction of eukaryotic expression vector of truncated LMP1 and the identification of its osteoinductive activityThe fragment of LMP1 (from the first amino acid to the 133th amino acid) is needed for LMP1's osteoinductive activity. Based on this, we designed and constructed eukaryotic expression vector of truncated LMP1. In this truncated LMP1, the LIM domain was deleted from the carboxyl terminal of the full length LMP1. Marrow stroma cells were transfected by the constructed eukaryotic expression vector. Forty-eight hours later a fresh medium containing 0.5 mg/ml G418 was added to replace the medium. The medium containing 0.5 mg/ml G418 was refreshed every three days. At day 6, 10 and 14 after cells were screened, cells were collected and the total RNA was extracted by Trizol method. tLMP cDNA was acquired by RT-PCR using the primers designed and synthesized on the basis of the gene sequence of tLMP. It was found that the level of tLMP mRNA was raised obviously compared with the levels of LMP1 mRNA of the blank control group and the transfection control group. After cells were transfected and screened, the alkaline phosphatase activities were detected and the cells were stained by Von Kossa method. The increase of the alkaline phosphatase activities and the formation of calcium nodus showed that transfection of the eukaryotic expression vector of truncated LMP1 could induce MSCs to differentiate to osteoblasts. Through the examination of the mRNA level of BMP2, we found that after transfection, the level of BMP2 mRNA was raised significantly. It was proved that tLMP has the similar function to full length LMP1.Section 3: Establishment of the technological platform for protein transductionProtein transduction domain can transduce proteins fused with them into cells without impairment to host cells. The proteins which are transduced into cells by PTD will keep their bioactivity in the cells. In order to transduce tLMP into cells, we established a technological platform for protein transduction. Based on the amino acid sequence of TAT peptide, we designed gene sequence which was suited to expression in prokaryocytes. We cloned the gene sequence, His-tag and multiple clone sites into the initial vector pET28a. At last, we obtained the prokaryotic expression vector pET28a-TAT.In order to test the expression capability of the prokaryotic expression vector pET28a-TAT and the protein transduction ability of TAT, the enhanced green fluorescent protein (EGFP) was cloned into the vector and fused with TAT. The recombinant vector was transformed into the host bacterial Escherichia coli ER2566. The expression of the fusion protein was induced by the addition of IPTG to the bacterial culture. The fusion protein was purified by affinity chromatograph. After being purified, the protein was used to co-incubate with MSCs for 5min. EGFP fluorescence was detected in MSCs using the fluorescence microscope. This experiment confirmed that TAT can transduce the proteins fused with it into cells quickly. Using this technological platform, we can transduce tLMP into cells with bioactivity.Section 4: Construction of prokaryotic expression vector and expression and purification of TAT-tLMP fusion proteinBased on the amino acid sequence of tLMP, we designed gene sequence which was suited to expression in prokaryocytes using molecular biological software Vector NTI. After that, the primers were designed and synthesized and the optimized tLMP gene was obtained by PCR. And then, the gene was cloned into prokaryotic expression vector pET28a-TAT. Finially, we got the recombinant vector pET28a- TAT- tLMP. The recombinant vector was transformed into the host bacterial Escherichia coli BLR (DE3). The expression of the fusion protein was induced by the addition of IPTG to the bacterial culture. The characteristic of fusion protein was analyzed and predicted using software Vector NTI. According to the result, a strategy for protein purification was proposed. The production mainly existed as inclusion bodies. In order to purify the fusion protein, host bacteria were collected and the inclusion bodies were purified. After that, the inclsion bodies were lysed by prepared buffer with 8M urea, so the fusion protein could be solubilized from inclusion bodies. Then, through two steps of purification, affinity chromatograph and ion exchange chromatography, we obtained the fusion protein with high purity.Section 5: Experiment of osteoinductive activity of TAT-tLMP fusion protein in vitro and in vivoIn this experiment, we examined the influence of the fusion protein on the proliferation, alkaline phosphatase activities and calcium nodus formation of MSCs in vitro. We also tested the ability to induce ectopia osteogensis of the fusion protein compounded with fibrin sealant. The proliferation of MSCs was not impacted by the fusion protein. The alkaline phosphatase activities of MSCs rose up significantly compared with control groups. After 14 days induction, we could find the formation of calcium nodus. All the results showed that the fusion protein could induce MSCs to differentiate to osteoblasts in vitro. Three weeks after the compounded material was implanted into thigh muscle pouches of the mice, there was no new bone or cartilage formation in experimental group, while new cartilage formed in induction control group ( FS+bBMP). According to this, we could conclude that the fusion protein has osteoinductive activity in vivo, but has no ability to induce ectopia osteogensis when used with FS.Section 6: Experiment of osteoinductive activity of Smurf1 WW domain interaction site peptide in vitro and in vivoIn the unique sequence of LMP1, there is a peptide which can bind to WW domain of Smurf1. The peptide synthesized artificially is able to compete with full-length LMP-1, Smad1 and Smad5 for binding with Smurf1. In this experiment, we examined the influence on the proliferation, the alkaline phosphatase activities and calcium nodus formation of MSCs in vitro of the peptides fused with TAT. Its capacity of ectopia osteogensis induction in vivo was also tested. Finally, we got the similar results to the previous experiment. The alkaline phosphatase activities of MSCs increased compared with control groups. We could also observe the formation of calcium nodus in experiment group. But unfortunately, the result of the experiment in vivo showed that the peptides could not induce ectopia osteogensis.Conclusion:1. LMP1 is a protein existing in cytoplasm, and the truncated LMP1 has same osteoinductive activity as the full length LMP1.2. TAT is a high-performance PTD which can transduce the protein fused with it into cells without changing its own bioactivity.3. The fusion peotein TAT-tLMP has osteoinductive activity, but its activity is weaker than rhBMP2'.4. The peptide of Smurf1 WW domain interaction site has the similar function as tLMP.5. When used in vivo experiment compounded with FS, neither the fusion protein TAT-tLMP nor the peptide of Smurf1 WW domain interaction site has the ability to induce ectopia osteogensis.