HYBRID MAGNETIC NANOPARTICLE-BASED AFFINITY PURIFICATION OF RECOMBINANT Rhodopseudomonas palustris COLLAGEN-LIKE PROTEIN

Abstract

Recombinant bacterial collagen-like proteins, particularly Rhodopseudomonas palustris collagen-like protein (RPCLP), represent a promising alternative to animal-derived collagens for pharmaceutical, cosmetic, and biomedical applications. This study investigates a novel purification strategy for histidine-tagged recombinant collagen-like protein (His-RPCLP) using functionalized magnetic nanoparticles in a hybrid affinity system, offering enhanced scalability and cost-effectiveness compared to conventional column-based affinity chromatography. The methodology encompassed three key stages: synthesis and functionalization of magnetic nanoparticles with metal-chelating ligands, development of a hybrid magnetic affinity purification protocol, and comprehensive characterization of the purified protein. Protein quantification was performed using Bradford assay, while purity assessment utilized sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with ImageJ®-based densitometric analysis. Process optimization was achieved through Face Centered
Central Composite Design (FCCCD) under Response Surface Methodology (RSM) framework, targeting simultaneous maximization of protein yield and purity. The experimental design focused on two critical parameters: functionalized magnetic nanoparticle loading and imidazole concentration in the elution buffer. Thirteen experimental runs were systematically evaluated to establish optimal operating conditions. Initial screening experiments demonstrated successful nanoparticle functionalization with an average recovery of 0.0418 g, yielding 0.39 mg/mL of purified His-tagged protein at 26% purity. Statistical optimization revealed optimal conditions producing a maximum RPCLP yield of 0.314 mg/mL with significantly improved purity of 91.51%, representing a 3.5-fold enhancement in protein purity compared to non-optimized conditions. However, ANOVA analysis indicated statistical insignificance of the optimization models (p > 0.05), suggesting the need for expanded experimental design incorporating additional factors or extended parameter ranges in future investigations. Despite model limitations, this study successfully demonstrates the feasibility of magnetic nanoparticle-mediated affinity as a viable purification platform for non-enzymatic recombinant proteins.