O Processing Of The Recombinant Protein

Processing the fermentation contents to isolate a recombinant protein is often a difficult operation, requiring as much art as science. In the fermentation broth are whole bacterial cells, lysed cells, cellular fragments, nucleotides, normal bacterial proteins, the recombinant protein, and particulate medium components. If a Gram-negative bacterium such as E. coli has been used, lipopolysaccharide endotoxins (pyrogens) may be present. When animal cell cultures are used, it is commonly assumed that virus particles may be present. Viruses can also be introduced by the culture nutrients, generated by an infected cell line, or introduced by animal serum. Purification of an rDNA protein while maintaining the factors that keep it in its active three-dimensional conformation from this mixture may be difficult, because each step must be designed to ensure that the protein remains intact and pharmacologically active. Assays must be designed that allow the activity of the protein to be assessed at each purification step. Consequently, the structure and activity of the recombinant protein must be considered at all stages of purification, and assays must be conducted to measure the amount of purified, intact protein.

A general scheme for purification of an rDNA protein is as follows95:

• Particulate removal. Particulates may be removed by cen-trifugation, filtration, ultrafiltration, and tangential flow filtration. Virus particles may be inactivated by heating if the rDNA peptide can tolerate the procedure.

• Concentration. The volume of the mixture is reduced, which increases the concentration of the contents. Often, concentration is achievable by the filtration step, especially if ultrafiltration is used.

• Initial purification. The initial purification of the mixture is sometimes accomplished by precipitation of the proteins, using a slow, stepwise increase of the ionic strength of the solution (salting out). Ammonium sulfate is a typical salt that can be used in cold, aqueous solutions. Water-miscible organic solvents such as trichloroacetic acid and polyethylene glycol (PEG) change the dielectric constant of the solution and also effect precipitation of proteins.

• Intermediate purification. In this stage, the proteins may be dialyzed against water to remove salts that were used in the precipitation step. Ion exchange chromatography is used to effect a somewhat crude separation of the proteins based on their behavior in a pH or salt gradient on the resin. Another step that may be taken is size exclusion (gel filtration) chromatography. Gels of appropriate molecular weight cutoffs can yield a somewhat low-resolution separation of proteins of a desired molecular weight. If a native bacterial protein that has been carried this far is nearly the same molecular weight as the rDNA protein, no separation will occur.

• Final purification. Final purification usually involves the use of high-resolution chromatography, typically highperformance liquid chromatography. An abundance of commercial stationary phases allows various types of adsorption chromatography (normal and reversed phase), ion exchange chromatography, immunoaffinity chromatogra-phy, hydrophobic interaction chromatography, and size exclusion chromatography. The protein fractions are simply collected when they elute from the column and are concentrated and assayed for activity.

• Sterilization and formulation. This step can be accomplished by ultrafiltration to remove pyrogens or by heating if the protein can withstand this. Formulation might involve reconstitution into stable solutions for administration or determining the optimum conditions for stability when submitting for clinical trials.

Complicating factors include (a) proteins unfolding into an inactive conformation during processing (it may not be possible to refold the protein correctly) and (b) proteases that are commonly produced by bacterial, yeast, and mammalian cells, which may partially degrade the protein.

Was this article helpful?

0 0
Healthy Sleep

Healthy Sleep

A Guide to Natural Sleep Remedies. Many of us experience the occasional night of sleeplessness without any consequences. It is when the occasional night here and there becomes a pattern of several nights in arow that you are faced with a sleeping problem. Repeated loss of sleep affects all areas of your life The physical, the mental, and theemotional. Sleep deprivation can affect your overall daily performance and may even havean effecton your personality.

Get My Free Ebook


Post a comment