Proteins & Peptides

Protein Structure

Protein concentration is an issue that the biological sciences have been dealing with for years. Many aspects of the study of proteins and exploitation of their therapeutic value requires concentration before and after processing. Each process step can range in scale from setting up a sitting drop of 100nl in microtitre plate based crystallization study to purification of the product from 10,000L fermentation. All of the currently available tools have their draw-backs whether it is from yield loss on membranes to dilution from capture columns.

One of the most commonly used techniques at all scales is membrane concentration. At the small scale this is usually based on concentrators that fit in a centrifuge or small pressure based filers both of which rely on dead-end filtration to push the supernatant through a membrane and retain the protein. The small scale methods used suffer badly from concentration polarization a phenomenon observed in membrane concentrations where the protein concentration at the membrane increases due to the removal of liquid, this is analogous to the cake formed in conventional filtration. In larger applications a cross flow membrane is commonly used where the pressure potential for filtration is provided by a tangential flow to the membrane surface.This cross flow helps to reduce the effect of concentration polarization by sweeping the membrane to clear the retained protein; however, due to constant solvent removal within the system there is still a polarization gradient setup at steady state. When a protein reaches ultra-high concentration at the membrane interface then gel formation is likely this can reduce the activity of the protein and, if the regime for re-suspension is not effective, lead to loss of yield. When we couple this to the non-specific binding effects of most membrane systems it makes this a technique that the industry is using for necessity rather than optimized performance.

One alternative for laboratory scale concentration is to use Centrifugal Vacuum concentration where the solvent is boiled from the supernatant under vacuum so that the temperature of boiling is below the denaturation threshold of the protein. This can dramatically improve both the yield after concentration and also the activity of the protein.This technique would not be used on the large scale but finds a niche in the small scale laboratory work, for example preparation of sample prior to screening against a panel of precipitants to determine the optimum conditions for crystallisation prior to XRD. For hanging and sitting drop experiments concentrations greater than 10 to 20 mg/ml are often required, this is very difficult to achieve in membrane based concentrators due to the limiting factors that have been highlighted above. Where the sample has been purified using reverse phase chromatography (typically peptides), leaving the sample in water and organic solvent, this cannot be concentrated using a membrane because the organic solvents damage the membrane. In a vacuum concentrator the sample remains in a micro-centrifuge tube and the volume remaining can be accurately determined by the time of concentration. One potential limitation of this technique is that the salt and buffer components of the solution will also be concentrated, hence this is ideally suited to be a polishing step that is used after a capture column or membrane concentration step and therefore allows the sample to reach the very high concentrations needed for crystallization. The most commonly used systems for these applications are the miVac range of biological concentrators or the EZ-2.

Peptide Synthesis

Peptide synthesis is normally performed using solid phase supported stub with the sequential addition of protected amino acids. Between each addition step, the protected terminal needs to be deprotected so that the next acid can react and join.  When the desired peptide sequence is complete, it is cleaved from the solid support, usually using a strong acid such as TFA.  The peptide in cleavage cocktail is then dried and purified. Following purification the peptide can be fast lyophilised in a Genevac HT series evaporator or dried using a traditional freeze drier.

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