A key challenge in recombinant protein production is to maintain and store the target protein in a soluble and stable form. Protein aggregation can compromise protein function and thus it is necessary to overcome this challenge to generate functionally active protein.

Aggregates can be categorised as either “insoluble” (able to be removed by centrifugation or filtration) or “soluble” (not easily separated from native protein). Techniques such as analytical size exclusion chromatography (SEC), dynamic light scattering (DLS) and ultracentrifugation play an important role in identifying soluble aggregates.

Aggregation can occur at any stage of the production pipeline:

  • Protein Expression (e.g. inclusion body formation)
  • Protein Purification
    • Cell lysis and extraction
    • Chromatography
    • Buffer Exchange
    • Concentration
    • Storage

A number of strategies can be employed to overcome aggregation and promote protein stability.

The use of fusion tags, such as maltose binding protein (MBP) or thioredoxin (Trx), can impart solubility on proteins expressed heterologously in E. coli. Modifying expression culture conditions (e.g. reducing temperature) may also improve solubility by promoting correct folding.

Buffer conditions can be optimised to improve the target protein’s solubility during purification. Additives such as reducing agents (e.g. ß-mercaptoethanol, DTT), chaotropes (e.g. urea, guanidium-HCl), kosmotropes (e.g. ammonium sulphate, glycerol), detergents (e.g. tween, CHAPS), amino acids (arginine, glutamine) and ligands or cofactors (protein-dependent) can be used in low concentrations to stabilise the target protein’s native structure. Additionally, buffer pH and ionic strength also influence protein stability. Therefore it is often necessary to screen an array of buffer conditions and additives to determine the optimal buffering environment for the target protein. Once these stabilising conditions are known, they can be implemented throughout the purification process.

High protein concentration can compromise protein stability. Consequently, it may be necessary to maintain a low protein concentration by increasing the sample volume during lysis and chromatography. In situations where a high final protein concentration is required, stabilising buffer components can be included to avoid protein aggregation and maintain solubility.

Many proteins are unstable at 4˚C for more than a few days, so the preferred strategy is to store purified protein at -80˚C. However, subjecting proteins to repeated freeze-thaw cycles often leads to protein precipitation, so it is good practice to scout stability in advance. Buffer conditions that favour protein solubility at 4˚C may not necessarily prevent aggregation during freeze-thaw. Glycerol is often added to the protein sample as a cryoprotectant.

Other practices that reduce the propensity for proteins to aggregate include:

  • Performing all purification steps at 4˚C
  • Minimising sample handling
  • Avoiding time delays between purification steps
  • Reducing exposure to air-liquid interfaces (e.g. by avoiding bubble formation)