Redesigning Nature: Developing a More Potent BMP2 Molecule for Expression in a Transgenic Puroindoline-Rice Expression System

Abstract

Bone Morphogenetic Protein 2 (BMP2) is a cytokine growth factor that elicits de novo bone formation in adult mammals. The use of BMP2 in surgical applications ranges from spinal fusion procedures to off-label uses such as dental implant augmentation. Currently, 1.5 mg/ml of BMP2 are necessary for these surgical procedures. The use of such relatively high concentrations of BMP2 leads to ectopic bone formation, provokes immune reactions hence rendering treatments ineffective and adds greatly to the overall expense of these therapeutic treatments. An engineered mutant BMP2 designed to have higher biological potency over the current wild type recombinant human BMP2 would reduce both dosages and costs in biomedical applications. The synthesis of a codon optimized DNA sequence designed for expression in rice would ensure high fidelity expression of such recombinant protein products in a biotech rice protein production platform. Although designed for rice recombinant protein expression, the codon optimized DNA sequence produces a fragment that corresponds to the theoretical fragment size of the C-terminal, mature monomeric peptide of BMP2 when expressed in E. coli. Results from in silico modelling of mutant BMP2 ligands docked with BMP receptors suggested that only certain mutations are tolerated at the L51 and D53 positions. Only certain mutants might have the same affinity for the receptor as the wild type due to steric interactions with other side chains on both the ligand itself and the receptor. For those mutants that did not possess steric conflicts, the recombinant L51-series BMP2 mutants produced a circular dichroism spectrum that was unique and differed from the spectrum of wild-type BMP2. C2C12 alkaline phosphatase activity assays of the wild-type BMP2 protein produced activity similar to previously published results, while the thirteen L51 substituted BMP2 mutant collection samples showed no bioactivity similar to a known negative activity mutant at this position. The expression potential of the codon optimized DNA BMP2 sequence in rice was calculated by comparing several computer generated sequences from online programs. Such models were used to assess whether the recombinant BMP2 possesses similar bioactivity to the BMP2 expressed in mammalian expression systems currently used.