Expression And Activity Evaluation Of Bone Morphogenetic Protein 10 And Its Mutants In CHO Cells

Bone morphogenetic protein(BMP10)plays an important role in the development of embryonic heart tissue and the repair of damaged myocardium in adults,and the evaluation of its drug-forming properties is a new hot topic in drug research.The natural BMP10 molecule containing 424 amino acid residues consists of 3 parts: signal peptide,lead peptide and maturation peptide.There is a Furin specific recognition site RIRR316 at the junction of the lead peptide and the maturation peptide.Furin cleavage causes heterogeneous expression products of recombinant BMP10,resulting in inconsistent analysis of BMP10 activity.Our previous study found that the presence of the lead peptide did not have a clear effect on the biological activity of BMP10,and expression of proBMP10 with the lead peptide may be more advantageous for pharmaceutical applications.In this paper,aim to provide a suitable BMP10 sample for its drug-forming studies,the mutants of proBMP10 were designed in Furin recognition site,a CHO cell expression system of mutants were established by site-specific integration,the biological activity of the mutants were analyzed.The main results are as follows:1.Two mutants of proBMP10,proBMP10-1(R313K)and proBMP10-2(R316K),were designed around the sequence of Furin cleavage of BMP10 at position 316,mutating arginine to lysine at positions 313 and 316 of proBMP10,respectively.Based on the site-specific integration technology platform in CHO cells constructed by our laboratory,donor plasmids EGFP-proBMP10,EGFP-proBMP10-1 and EGFP-proBMP10-2,with proBMP10,proBMP10-1 and proBMP10-2 genes with homologous sequences of CHO NW-003626341.1 locus,were constructed.The three donor plasmids were cotransfected with sg RNA plasmid and Cas9 plasmid to CHO-K1BAK-/BAX-(Ie3)cells with anti-apoptotic ability by CRISPR/Cas9 gene editing technology,respectively.Two cell lines of Ie3-proBMP10,two cell lines of Ie3-proBMP10-1 and one cell line of Ie3-proBMP10-2 were obtained by puromycin pressure sieving,flow cytometry sorting and series validation for successful targeted integration.2.Five recombinant Ie3 cell lines were domesticated in suspension and three cell lines,Ie3-proBMP-10,Ie3-proBMP10-1 and Ie3-proBMP10-2,were obtained for passaging culture.After fifty generations of passages,it was found that the three cell lines could stably express EGFP and target protein in different generations,which could meet the quality requirements for stable expression and expanded culture of the main cell bank and production cell bank.3.proBMP10 and its mutant protein were obtained by fed batch culture of three cell lines and Nickel column affinity chromatography.SDS-PAGE revealed that the relative molecular masses of proBMP10 and proBMP10-1 were 58 k Da and 45 k Da,and that of proBMP10-2 was58 k Da.Immunoblotting using the corresponding antibodies proved that the purified products were the target proteins.Furin digestion experiments in vitro showed that the band at 58 k Da of proBMP10-2 and proBMP10-1 disappeared and the band at 45 k Da was retained,while proBMP10-2 still showed a single band at 58 k Da,indicating that proBMP10-2 was not cleaved by Furin,while proBMP10-1 was still cleaved by Furin.4.In vitro experiments using mouse teratoma cells P19 revealed that proBMP10 and its mutants proBMP10-1 and proBMP10-2 had some biological activity before and after Furin digestion.Compared with the negative control,proBMP10,proBMP10-1 and proBMP10-2 all stimulated Smad6 expression in P19 cells to some extent.It is suggested that proBMP10-2 is biologically active,verifying that the designed proBMP10 mutant proBMP10-2(R316K)is no longer cleaved by Furin and still had biological activity after mutation.