Protein Expression and Purification Protocol: Practical Steps

Producing high-quality proteins is essential for biochemical research, structural biology, and therapeutic development. A well-optimized protocol ensures efficient expression and purification while maintaining protein integrity and functionality.

This guide outlines key practical steps in protein expression and purification, from selecting an appropriate system to analyzing the final product.

Expression Systems

Choosing an expression system is fundamental to producing recombinant proteins with the desired yield, solubility, and post-translational modifications. Each system has advantages and limitations depending on the protein’s characteristics and intended application.

Bacterial Expression

Escherichia coli is the most commonly used host due to its rapid growth, cost-effectiveness, and ease of genetic manipulation. Strong promoters such as T7 or lac drive high protein synthesis. However, bacterial systems lack post-translational modifications like glycosylation and disulfide bond formation, which can be crucial for function. Co-expression with chaperones or using specialized strains like BL21(DE3) can enhance solubility and folding. Inclusion body formation is a common challenge, requiring refolding protocols or fusion tags (e.g., GST or MBP) to improve solubility. Despite these limitations, bacterial expression is widely used for enzyme studies and high-throughput screening.

Yeast Expression

Saccharomyces cerevisiae and Pichia pastoris enable eukaryotic protein expression with post-translational modifications while maintaining relatively high yields. Yeast systems offer secretory expression, simplifying purification, and the ability to perform glycosylation, though it differs from mammalian patterns. P. pastoris is favored for its methanol-inducible AOX1 promoter, allowing tightly regulated expression. Strain selection (e.g., GS115 or KM71) and secretion signals like α-mating factor influence efficiency. Yeast provides a balance between cost and complexity, making it suitable for enzymes, antibodies, and therapeutic proteins.

Mammalian Expression

Mammalian cells, such as HEK293 and CHO, are used for proteins requiring native folding, complex glycosylation, or functional post-translational modifications. Transient transfection or stable cell line generation ensures controlled expression, with CMV and EF1α promoters commonly used. Serum-free suspension cultures improve scalability, while optimized media formulations enhance yield. Despite higher costs and slower growth, mammalian expression is indispensable for biologics, including monoclonal antibodies and therapeutic enzymes. Baculovirus-infected insect cells offer an alternative for large-scale production with improved folding and processing.

Vector Design

An effective expression vector is critical for high-yield, functional protein production. The vector integrates elements that regulate transcription, translation, and stability. Selecting the optimal vector involves balancing promoter strength, selection markers, and fusion tags while ensuring compatibility with the host system.

Promoter selection dictates transcription initiation and regulation. In bacterial systems, the T7 promoter is widely used for strong, inducible expression, while the arabinose-inducible pBAD promoter offers tunable control. Eukaryotic vectors use CMV for high-level expression or inducible systems like Tet-On/Tet-Off for regulation. Enhancer sequences, such as the SV40 enhancer in mammalian vectors, can further boost efficiency. Codon optimization tailored to the host improves translation fidelity.

Fusion tags enhance solubility and simplify purification. His6, GST, and MBP tags facilitate recovery via affinity chromatography while influencing folding and stability. His-tagged proteins allow rapid purification using nickel or cobalt resin. GST and MBP fusion partners improve solubility by acting as chaperones, reducing aggregation. Protease cleavage sites, such as TEV or thrombin, enable precise tag removal post-purification. Signal peptides direct secretory expression, with pelB for bacterial periplasmic targeting and α-factor for yeast secretion.

Induction Conditions

Optimizing induction conditions balances yield and solubility while preventing cellular toxicity. Timing and inducer concentration influence transcriptional activation, and excessive protein production can lead to misfolding or aggregation.

Temperature affects expression efficiency and solubility. Lowering induction temperature—from 37°C to 16°C or 25°C in bacterial systems—reduces aggregation by slowing synthesis, allowing proper folding. In mammalian and yeast systems, temperature shifts can influence post-translational modifications. The duration of induction varies, with bacterial expression typically requiring 3–6 hours, while eukaryotic hosts may need overnight incubation.

Inducer concentration must be carefully calibrated. In bacterial systems, IPTG activates T7 promoters, but excessive levels can cause metabolic stress. A range of 0.1–1.0 mM is typically used, with lower concentrations favoring soluble expression. Arabinose for pBAD systems allows more precise regulation. In yeast, methanol induction in Pichia pastoris must be carefully managed to prevent toxicity. Gradual adaptation to methanol feeding, starting at 0.5%, enhances expression while maintaining cell health.

Cell Lysis Techniques

Efficient cell lysis maximizes protein recovery while preserving structural integrity. The choice of lysis method depends on the host organism, protein localization, and downstream purification requirements.

Mechanical lysis methods, such as sonication and French press homogenization, are widely used for bacterial and yeast cells. Sonication applies high-frequency sound waves to shear membranes, but excessive use generates heat, risking denaturation. French press homogenization ruptures cells under high pressure, reducing thermal stress. These methods are effective for cytoplasmic proteins but may require additional steps for periplasmic or membrane-bound targets.

Chemical lysis relies on detergents and chaotropic agents. Non-ionic detergents like Triton X-100 and NP-40 maintain protein functionality, whereas stronger surfactants like SDS facilitate complete membrane solubilization but require careful removal before purification. Osmotic shock, using hypertonic buffers followed by rapid dilution, is frequently employed for periplasmic protein extraction, selectively releasing proteins while minimizing cellular debris.

Purification Methods

After expression and extraction, purification isolates target proteins from contaminants. The method depends on biochemical properties, expression system, and intended applications.

Affinity Chromatography

Affinity chromatography exploits specific interactions between a protein and its ligand. His-tagged proteins are purified using immobilized metal affinity chromatography (IMAC), where nickel or cobalt resin binds histidine residues for selective elution with imidazole. GST fusion proteins bind glutathione resin, while MBP fusions use amylose resin. The high specificity reduces contamination, but non-specific binding and metal ion leaching require optimization of wash and elution conditions.

Ion Exchange Chromatography

Ion exchange chromatography (IEX) separates proteins based on charge differences. Proteins interact with charged resins—anion exchangers like DEAE or cation exchangers like CM—depending on their isoelectric point. Gradually increasing salt concentration in the elution buffer differentially displaces proteins, refining purity. Buffer pH optimization ensures efficient binding. Combining IEX with affinity chromatography enhances purity, particularly for stringent contaminant removal.

Size Exclusion Chromatography

Size exclusion chromatography (SEC), or gel filtration, separates proteins by molecular weight, making it ideal for final polishing and buffer exchange. Proteins traverse a porous matrix, with larger proteins eluting first and smaller molecules experiencing greater retention. SEC effectively removes aggregates and small impurities, ensuring monodispersity for structural studies. Optimizing column parameters, such as pore size and flow rate, enhances resolution and prevents peak broadening.

Post-Purification Analysis

Assessing protein quality confirms purity, structural integrity, and functional activity. Without rigorous characterization, contaminants or misfolded proteins may compromise downstream applications.

SDS-PAGE and Western blotting remain fundamental for purity assessment and identity confirmation. SDS-PAGE resolves proteins by molecular weight, revealing degradation products or contaminants. Western blotting, using antibodies targeting specific epitopes or tags, provides additional validation, particularly for low-abundance proteins. High-performance liquid chromatography (HPLC) offers quantitative purity assessment, detecting minor impurities.

For structural characterization, circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) assess secondary structure and thermal stability, respectively. Dynamic light scattering (DLS) evaluates aggregation tendencies, ensuring proper folding. Functional assays, such as enzymatic activity measurements or ligand binding studies, confirm bioactivity. Mass spectrometry provides precise molecular weight determination, identifying post-translational modifications or truncations.