Folding constraints and engineering strategies for functional production of structurally complex phage receptor-binding proteins in Escherichia coli

Bacteriophage receptor-binding proteins (RBPs) provide highly specific recognition of bacterial hosts and are being developed as biorecognition molecules and targeting components in antibacterial applications. However, recombinant production in Escherichia coli is frequently limited by poor soluble recovery and uncertain preservation of native assembly and function. Among the structurally complex RBPs emphasized here, many characterized tail fibers, tailspikes and RBP-associated depolymerases form oligomeric, often trimeric, β-rich assemblies that can create protein-specific folding and assembly constraints. This review critically evaluates four candidate constraints: conformational instability, structural complexity associated with multidomain and β-rich architectures, transient exposure of aggregation-prone surfaces, and imbalances between protein synthesis and productive folding or assembly. We assess domain and construct engineering, fusion partners, cognate chaperone co-expression, and modulation of induction kinetics. The proposed framework draws on experimental evidence from model RBPs and construct-specific studies. It also incorporates findings on other phage structural proteins and general principles of protein folding. Greater soluble recovery alone does not establish native oligomerization, structural integrity, stability or receptor-binding activity. Accordingly, the proposed structure-folding-function framework is hypothesis-generating rather than a universally validated mechanism or predictive algorithm. Based on current evidence, we suggest that poor soluble production of selected structurally complex RBPs reflects interacting, protein-specific folding and assembly constraints rather than a single universal defect. Optimization remains largely empirical because no prospectively validated framework currently links sequence and structural features to suitable expression configurations. Integrating standardized screening, quantitative folding and assembly measurements, and structure-informed prediction may support mechanism-guided strategy selection, provided that soluble recovery is verified by native assembly, stability, receptor binding, or another predefined application-relevant function. Overall, this review evaluates experimentally supported and proposed contributors to RBP insolubility during recombinant expression and summarizes potential strategies for improving soluble and functional production.