The Preparation Of A New Thrombopoietic Fusion Protein Through Gene-engineering Approach And Study On The Underlying Mechanism Of Its Actions

With the development of society, thrombocytopenia caused by various reasonsbecomes more and more common. The megakaryocyte (MK) is a bone marrow cellresponsible for the production of platelets. Thrombopoiesis are ligations of the cytoplasmfrom megakaryocytes, can be promoted effectively by regulating the proliferation anddifferentiation of MKs. Therfore, considerable attention has been devoted togene-engineering products of hematopoietic growth factors which could promote plateletsproduction. However, these products for thrombocytopenia treatment in clinic are scarce atthe present stage, and the exisiting products have notable disadvantages, such as theproduction of neutralizing antibodies, prominent side effects and expensive costs. In all,there is an urgent demand for new drugs which can promote thrombopoiesis efficiently.Thrombopoietin(TPO), which was previously termed as MK growth and developmentfactor(MGDF), is the most important cytokine which regulates megakaryocytopoiesis andthrombopoiesis. Through binding to its receptor(c-Mpl), TPO activates a cascade of signaltransduction pathways which promote MKs proliferation and differentiation. Accordingly,recombinant human thrombopoietin(rhTPO) and pegylated recombinant humanmegakaryocyte growth and development factor(PEG-rhMGDF) as the first generationthrombopoietic growth factors have been developed through gene-engineering approach.However, the emergence of autoantibody against PEG-rhMGDF, which could cross-reactwith and neutralize endogeous TPO, results a total ban on clinical trials of recombinanthuman TPO gene-engineering products by the U.S. FDA. Thrombopoietin mimeticpeptide(TMP), which was identified by phagy display technology, is a14-mer peptide withhigh affinity to c-Mpl and shares no homologous sequences with TPO. Studies showed thatTMP could promote MKs proliferation in vitro, but not induce TPO neutralizing antibody invivo. Nevertheless, TMP is hardly to be prepared by gene-engineering methods due to its small molecular weight, and it is easy to be degraded in the circulation, which together limitits application.Human growth hormone(hGH) has been reported to play an important role inproliferation and differentiation of hematopoietic and megakaryocytic progenitor cells, andenhance thrombopoiesis. hGH exerts its function mainly by binding to GH receptor(GHR),a kind of type I cytokine receptor as c-Mpl, and in turn activates the proliferation anddifferentiation related signal pathways. Therefore, we envisaged to construct a chimericprotein which fused TMP to hGH. On one hand, fusion with hGH increases molecularweight, which makes preparation of TMP through genetic engineering approach possibleand extends its half life in vivo; on the other hand, by fusion with hGH, there may be asynergetic effect on megakaryocytopoiesis and thrombopoiesis.Based on our previous works, with funding of national “863” plan and major scienceand technology poject of new drug development, the chimeric protein rTMP-GH was triedto prepared by expression and purification in Pichia pastoris. In order to improve thebioactivity of the fusion protein, a chimeric protein containing a tandem dimmer of TMP(dTMP) fused to hGH was then constructed in Escherichia coli with pMAL-p2x expressionvector. After identifying its significant effect on promotion megakaryocytopoiesis,dTMP-GH was expressed in inclusion body form through temperature control inducing inEscherichia coli, which simplifies the preparation process and improve the yield, Byoptimizing the technology, high densty fermentation and production amplification of thefusion protein were successfully achieved. Finally, a gene-engineering product ofdTMP-GH with high purity over than98%and fuction of promoting the proliferation anddifferentiation efficiently was obtained. At the same time, the mechanism of dTMP-GHpromoting the proliferation and differentiation of MKs was discussed from the perspectiveof signaling pathways. The main results and conclusions are summarized as follows:1. The recombinant eukaryotic expression plasmid pPIC9K-rTMP-GH was constructedsuccessfully by genetic recombination. The His+/Mut+polyclonal expression strain withhigh G418resistance was screened out, followed by the expression of rTMP-GH throughhigh density fermentation in Pichia pastoris GS115. The recombinant fusion proteinrTMP-GH with purity over than95%was then obtained by purification with ultrafiltration,anion column and molecular sieve. A certain activity on promoting MKs proliferation of rTMP-GH was confirmed in vitro.2. By rational designed restriction sites, the recombinant expression plasmidpMAL-dTMP-GH was constructed successfully. After electrically transformation toEscherichia coli, the soluble fusion protein MBP-dTMP-GH was obtained through IPTGinduction in positive clone. By application of amylase resin chromatography, Xa factordigestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusionprotein with purity of98.5%and without redundant amino acid at N terminal was obtained.The functional assays in vitro showed that dTMP-GH could promote the proliferation ofmegakaryoblast cell line M07e in a dose-dependent manner. Moreover, an enhanced effectof dTMP-GH was found as compared with equimolar concentration of rTMP-GH fusionprotein.3. By using of prokaryotic expression vector pBV220, the expression of dTMP-GHthrough high densty fermentation in inclusion body form was achieved successfully, withthe yield of500mg/L. A purification strategy including inclusion body denaturation,refolding, cation chromatography, anion chromatography and molecular sievechromatography was established to obtain dTMP-GH with purity over than98%andpencent recovery of20%. There was no significant difference in the bioactivities ofdTMP-GH obtained through the above two approaches.4. The MKs proliferation activity of dTMP-GH was significantly stronger thanequimolar concentration of dTMP and obviously weaken by GH antagonist Pegvisomant,while there is no notable effect on MKs proliferation of rhGH. It suggested that hGH mayenhanced the effect on promoting MKs proliferation and differentiation of dTMP by GHRmediated.5. Result of Western blot analyse displayed that the phosphorylation levels of STAT5and ERK1/2in M07e cells were up-regulated in15min and lasted to1h by dTMP. Therewas no detectable promotion of STAT5phosphorylation after rhGH treatment, but itinduced obviously phosphorylation of ERK1/2in1h and continuing for more than3h. Itsuggested that the proliferation and differentiation of MKs induced by dTMP may beenhanced by rhGH through slow and sustained activation of ERK1/2signaling pathway.6. Confocal laser scanning and Western blot detection showed that rhGH treatmentup-regulated the expression of actin in mature megakaryocytic cell line Meg-01, in turn promoted myoneme stretching and pseudopodia extending. It indicated that rhGH has thefunction of promoting MKs maturation. Further research in Meg-01revealed that rhGHup-regulated the phosphorylation level of Akt significantly, which was not activatedobviously by dTMP. It suggested that rhGH promote MKs differentiation and maturationthrough the activating of Akt signal pathway.7. The fusion protein dTMP-GH could exert bioactivites of both dTMP and rhGH. Thephosphorylation level of ERK1/2in M07e was up-regulated markedly and lasted more than3h by the treatment of dTMP-GH. And Pegvisomant was able to inhibited the sustainedactivation of ERK1/2by dTMP-GH. In Meg-01, the role of dTMP-GH was to up-regulatethe phosphorylation level of Akt, which could be inhibited by Pegvisomant too.8. Evidence on human CD34+derived primary megakaryoctes cultured for7dconfirmed that dTMP-GH was able to up-regulated STAT5and ERK1/2phosphorylationlevel, and the phosporylation of ERK1/2sustained for over3h. Additionally, Akt signalpathway in cells cultured for11d could be activated by dTMP-GH.In this study, by exploring different expression systems, optimizing expressionconditions and purification processes, we successfully fulfilled the efficiently expressionand preparation of a recombinant fusion protein that constructed with a tandem dimmerTMP fused to rhGH in Escherichia coli. A fusion protein of dTMP-GH with high purityover than98%was finally obtained. Experiments in vitro confirmed that dTMP-GH had theactivity of promoting the proliferation and differentiation of MKs. The major effect ofdTMP was to promote the proliferation and differentiation of early MKs, while GHenhanced the activity of dTMP on promoting MKs proliferation, as well as promoteddifferentiation and maturation of MKs. Therefore, the fusion of hGH not only increased themolecular weight of dTMP, which made it possible to be prepared by gene-engineeringapproach, but also had prominent effects on promoting proliferation, differentiation andmaturation of MKs. The novel thrombopoietic fusion protein, dTMP-GH, showed awonderful prospect for the development and application.