Optimized expression and purification of a soluble BMP2 variant based on in-silico design.

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

Expression and purification of soluble HIV-2 viral protein R (Vpr) using a sandwich-fusion protein strategy.

Viral accessory proteins of the human immunodeficiency virus (HIV), including virus protein R (Vpr), are crucial for the efficient replication of the virus in the host organism. While functional data are available for HIV-1 Vpr, there is a paucity of data describing the function and structure of HIV-2 Vpr. In this report, the construction of a His6-MBP-intein1-Vpr-intein2-Cyt b5-His6 fusion protein is presented. Unlike previous research efforts where only microgram quantities of HIV-1 Vpr could be produced, this construct enabled soluble milligram yields via an Escherichia coli over-expression system. Straightforward protein purification of HIV-2 Vpr was achieved by standard chromatography routines and autocatalytic intein cleavage. Preliminary structural studies by circular dichroism (CD) and NMR spectroscopy revealed that the protein is stable in the presence of micellar concentrations of the detergent DPC and adopts an α-helix secondary structure.

Enhanced selective cellular proliferation by multi-biofunctionalization of medical implant surfaces with heterodimeric BMP-2/6, fibronectin, and FGF-2.

Increasing cell adhesion on implant surfaces is an issue of high biomedical importance. Early colonization with endogenous cells reduces the risk of bacterial contamination and enhances the integration of an implant into the diverse cellular tissues surrounding it. In vivo integration of implants is controlled by a complex spatial and temporal interplay of cytokines and adhesive molecules. The concept of a multi-biofunctionalized TiO2 surface for stimulating bone and soft tissue growth is presented here. All supramolecular architectures were built with a biotin-streptavidin coupling system. Biofunctionalization of TiO2 with immobilized FGF-2 and heparin could be shown to selectively increase the proliferation of fibroblasts while immobilized BMP-2 only stimulated the growth of osteoblasts. Furthermore, TiO2 surfaces biofunctionalized with either the BMP-2 or BMP-2/6 growth factor and the cell adhesion-enhancing protein fibronectin showed higher osteoblast adhesion than a TiO2 surface functionalized with only one of these proteins. In conclusion, the presented immobilization strategy is applicable in vivo for a selective surface coating of implants in both hard and connective tissue. The combined immobilization of different extracellular proteins on implants has the potential to further influence cell-specific reactions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2910-2922, 2018.