Recombinant Protein Expression and Purification in the Era of Artificial Intelligence: Bridging Classical Biochemistry and Molecular Engineering

Recombinant protein production has evolved from an empirical biochemical procedure into a predictive engineering discipline that brings together molecular genetics, host biology, biophysics, synthetic biology, and artificial intelligence. The production of proteins, particularly complex classes such as membrane receptors, multidomain assemblies, intrinsically disordered regulatory proteins, and glycoproteins, demands expression environments that synchronize transcriptional load, translational kinetics, folding trajectories, and post-translational modifications (PTMs). Advances in synthetic biology, coupled with omics-guided host engineering, enable fine-tuning of several of these interconnected processes. In parallel, cell-free protein synthesis platforms offer programmable, decoupled environments that enable the production of structurally intricate, aggregation-prone, or cytotoxic proteins. These developments reposition recombinant protein science as a cohesive, predictive, and highly tunable platform capable of producing proteins with high structural integrity and functional fidelity. Despite remarkable technological progress, a cohesive framework that integrates host selection, construct engineering, purification strategies, and computational design around the specific requirements of the target protein has remained elusive. Here, we present a protein-centric perspective on recombinant protein production, linking the molecular determinants of folding, translational kinetics, PTMs, and cellular stress responses to rational decisions throughout the production pipeline. By synthesizing advances in expression platform engineering, purification technologies, biophysical quality assessment, artificial intelligence, synthetic biology, and systems-level optimization, we highlight how recombinant protein production is evolving from a largely empirical practice into a predictive engineering discipline. We further discuss unresolved challenges associated with structurally complex proteins and outline emerging opportunities that are poised to redefine the future of protein biomanufacturing, structural biology, and therapeutic development.