Protein Expression
A number of proteins are isolated from microorganisms.
These can be cultured and isolated either by batch culture or continuous culture.
Foreign genes can be expressed as protein in bacterial systems.
The construct must have a bacterial promoter, ribosomal binding site, and a transcription terminator.
The promoter plays a critical role in protein expression. Promoters can be constitutive or regulated, strong or weak. The choice of a promoter is dependent on how the foreign protein interacts with the bacterial cell. Some foreign proteins are toxic to the bacteria when they accumulate to high concentrations. Others will form insoluble aggregates. Some are toxic when expressed in even very small quantities. The question then becomes, which promoter will produce the most protein that is in a usable form?
Over production of a protein or improper disulfide bond formation can lead to insoluble proteins forming inclusion bodies.
Question:
Describe the type of promoter that would be appropriate for proteins
a, b, and c in the plot below.
There are a number of inducible promoters in E. coli.
Gene Fusion Proteins
Sometimes expression of a protein as part of a gene fusion can assist in keeping the foreign protein soluble or it may aid in the secretion of the protein or in its isolation.
Insertion of a foreign gene into a fusion cassette requires that the reading frames are matched during the construction.
The bacterial portion of the fusion product may contain signals for secretion of the peptide product into the extracellular space with eventual passage into the culture media.
Question:
If a fusion protein was secreted into the media, which form of culturing
the cells would be most efficient, batch culturing or continuous culturing?
The bacterial portion of the fusion product may contain signals for efficient isolation, for example by affinity chromatography.
Fusion protein products can be designed so that the bacterial portion of the fusion peptide can be cleaved from the foreign peptide either by a specific protease site, or a chemical cleavage site.
Question:
Once the bacterial peptide is cleaved from a protein purified by affinity
chromatography, how would you separate the bacterial peptide from the
protein product?
Design and potential problems with expressing foreign proteins in E. coli.
In designing expression clones, there are several items that must be considered in order to get a successfully producing clone.
Additionally, E. coli can not synthesize disulphide bonds, because it is generally a reducing environment.
Also, E. coli may have specific proteases that rapidly degrade the target protein.