Comparison of Recombinant Protein Expression Systems: Process Selection from Prokaryotic to Eukaryotic

Expression systems affect key properties of recombinant proteins

The biological function of a recombinant protein depends not only on its amino acid sequence, but also on disulfide bond pairing, glycosylation, phosphorylation, spatial folding, and aggregation state. Therefore, the choice of expression system is the first critical decision in recombinant protein development. Prokaryotic systems generally feature rapid growth, relatively low culture cost, and easier achievement of higher yields, but they lack complete eukaryotic post-translational modifications, and some complex proteins may be expressed as inclusion bodies, requiring refolding steps; eukaryotic systems are closer to native proteins in folding and modification, but culture cycles and process complexity are usually greater.

Comparison of prokaryotic and eukaryotic expression systems

Escherichia coli is a common prokaryotic expression host and is suitable for recombinant protein fragments, cytokines, or enzymes with relatively simple structures and no need for complex glycosylation. Yeast expression systems combine the advantages of single-cell growth with partial eukaryotic modification capability and are often used for secretory expression and proteins with certain glycosylation requirements. Insect cell-baculovirus systems can perform more complex folding and post-translational modifications and are suitable for the expression of virus-like particles, membrane proteins, and multi-subunit complexes. Mammalian cell systems are more suitable for recombinant proteins with high requirements for native conformation and glycoforms, but expression cycles and costs are relatively higher. The specific choice should be comprehensively evaluated in light of the target protein structure, immunoreactivity requirements, production capacity planning, and downstream purification costs.

Key control points in upstream processes

At the gene sequence design stage, codon optimization, signal peptide screening, promoter strength, and fusion tag position may all affect soluble expression and protein stability. During culture, temperature, pH, dissolved oxygen, feeding rate, and induction timing need to be controlled to avoid excessive accumulation of metabolic byproducts. For inclusion body expression, washing, denaturation, and refolding conditions need to be systematically optimized; for secretory expression, attention should be paid to protein degradation and aggregation in the culture supernatant. It is generally recommended to establish evaluation methods for expression level, soluble proportion, and activity recovery early in order to rapidly screen clones and culture conditions.

Scalability and quality consistency

When scaling up from laboratory shake flasks to fermenters or bioreactors, mixing efficiency, dissolved oxygen gradients, shear force, and temperature distribution will change, potentially affecting the quality attributes of the recombinant protein. It is recommended to use a combination of stepwise scale-up and scale-down models to confirm the ranges of critical process parameters. Process analytical technology can help monitor culture status in real time and reduce batch-to-batch variation. Ultimately, good process characterization and stable raw material sources are the foundation for ensuring batch-to-batch consistency of recombinant proteins and also provide more reliable intermediates for subsequent purification and formulation development.