Dissolube Expression And Purification Of RTMP-GH In Escherichia Coli And Studies Of Its Effects On Thrombocytopoiesis

Nowadays thrombocytopenia is frequently seen in clinic. However, there are few effective drugs for this kind of disease. Neotypes of drugs with high effective thrombocytopoiesis are expected.Thrombopoietin (TPO), which is also called megakaryocyte growth and development factor (MGDF) or thrombopoietic-stimulating factor (TSF), has been considered to be the principal regulator of megakaryopoiesis and platelet production. The effects of TPO on megakaryocytopoiesis and thrombocytopoiesis are mediated by its receptor, c-Mpl, expressed on the surface of megakaryocyte and megakaryoblast cells. Because there was a risk of producing neutralized antibody of TPO for pegylated recombinant human (rh) MGDF used in vivo, rhTPO can not be proofed by FDA in U.S.A until now. Recently, a small peptide consisted of 14 amino acids was reported to bind c-Mpl with high affinity and has functions in stimulating proliferation and differentiation of megakaryocytes as same as TPO, while it is heterogenous to TPO, so that it was called TPO-mimetic peptides (TMP). Because of small size, TMP is difficult to be produced by gene engineering and easily degraded in blood circulation.In the last few years, human growth hormone (hGH) was found to have significant effects on megakaryocytopoiesis and thrombocytopoiesis by acting primarily on megakaryocytes and megakaryoblast cells. However, because hGH has extensive physiological functions inside the body, its availability ratio in promoting megakaryocytopoiesis and thrombocytopoiesis is relatively low. Therefore, we set up a plan of fusing TMP to hGH through gene recombination technique. By fusion, hGH will be targetedly delivered to megakaryocyte /megakaryoblast cells with the guide of TMP, on the other hand, TMP can be easily obtained through gene engineering by fusion to hGH.In this study, we first subcloned the cDNA coding for rTMP-GH into the expression vector pMAL-p2x. By fusion to MBP, rTMP-GH was efficiently expressed in the cytoplasm in E. coli. After Xa factor digestion and purification, the recombinant protein TMP-GH was finally obtained. Then, the effects of rTMP-GH on megakaryocytopoiesis and thrombocytopoiesis were identified by a series of experiments performed in vitro and in vivo. The main results and conclusions are summarized as follows:1. The cDNA coding for TMP-GH was subcloned into the expression vector pMAL-p2x by PCR amplification, restriction enzyme digestion, ligation, and transformation. By induction with 0.1mol/L IPTG at 25℃overnight, the MBP-TMP-GH was highly expressed in the cytoplasm. After removal of MBP with Xa factor digestion followed by anion exchange chromatography and gel filtration, the purified rTMP-GH was successfully obtained (protein purity up to 95%).2. SDS-PAGE revealed that molecular size of the purified protein was about 24KD, which is coincident with the theoretic size of rTMP-GH. Western blot analysis with hGH antibody further confirmed that the purified protein was rTMP-GH.3. The results of cell adhesion experiments showed that rTMP-GH could bind to megacaryocyte cultured in vitro, suggesting that TMP functions well in the fusion protein. Megakaryocyte colony formation unit counting and MTT assay revealed that rTMP-GH had significant effects on megakaryocyte proliferation compared with single hGH and TMP.4. Western blot and RT-PCR detection showed that rTMP-GH could up-regulate the expression of transcription factor GATA-1 significantly in cultured megakaryocytes. These results further suggested that rTMP-GH have significant effects on megakaryocytopoiesis and thrombocytopoiesis.5. The results of animal experiments showed that the platelet number in peripheral blood was reduced significantly and reached to the nadir around 16 days in Balb/c mice received 5Gyγray irradiation. After irradiation, 400μg/kg of rhGH, rTMP-GH, or rhIL-11 was subcutaneously injected to the injured mice every other day。It was found that rTMP-GH treatment resulted in more significant increases in blood platelet than rhGH and rhIL-11. Bone marrow biopsy results showed that rTMP-GH treatment could dramatically ameliorate the reconstruction of bone hematopoiesis, especially for megakaryocytopoiesis.Conclusions: In this study, we successfully fulfilled the expression and prepration of a recombinant fusion protein, rTMP-GH, through gene engineering in E. coli. Both in vitro and in vivo experiments demonstrated that rTMP-GH had significant effects on megakaryocytopoiesis and thrombocytopoiesis, suggesting that this fusion protein can be used as an effective agent for the treatment of thrombocytopenia.