Considerations for Minimizing Aggregation

Proteos Chromatography Fraction Collector

Proteos is a US-based, employee-owned contract research organization providing custom, high-quality recombinant protein production services to the life-science community.  Powered by a team of scientists with diverse expertise, Proteos produces hundreds of recombinant proteins per year that meet the highest purity standards crucial for drug discovery and structural biology research.

Previously, the importance of protein solubility during the production process was discussed. Below we continue with considerations that can improve protein solubility and reduce aggregation.

Bacterial cells are a popular choice for recombinant protein expression due to the low production costs. Unfortunately, the complex structure of many eukaryotic proteins renders them insoluble in bacteria. Although it is possible to recover the protein of interest from inclusion body aggregates, it is frequently improbable.

Typically, eukaryotic proteins exhibit improved solubility when expressed in insect or mammalian cells. Further optimization of the cell line, growth parameters, and harvest times will also improve solubility and minimize aggregation during recombinant production.

To stabilize the protein and reduce aggregation during purification, buffer excipients, including salts, sugars, glycerol, detergents, or reducing agents are often useful.  Selecting buffers with the optimal pH is also critical since proteins are less soluble at their isoelectric point (pI) when the net charge is neutral. In addition to buffer formulation, efficient workflows that expedite purification can reduce the chances of aggregate formation. Purification should be carried out at a low temperature, and accidental heating of the sample should be avoided during steps such as lysis and centrifugation. Furthermore, if an unstable protein becomes too concentrated, it is more likely to aggregate. To minimize aggregation and sample loss, the protein may need to be maintained at a lower concentration during purification and preparation for long term storage.

Customized milestone-driven project designs are the cornerstone of data-driven workflows that lead to higher probabilities of project success. The Proteos team will work collaboratively with you to develop an application-focused scope of work. Contact us today to discuss your protein production needs. 

Tags: Solubility

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