Recombinant protein production by filamentous fungi
Filamentous fungi are single-celled eukaryotic organisms. The fungi used in protein production are Generally Regarded As Safe (GRAS) and subjected to extensive study. They grow rapidly on cheap media in fermenters, although not as quickly as certain prokaryotes like Escherichia coli. They can produce homologous proteins such as cellulases at levels, as high as 40g/l and secrete them into the medium for simple and cheap separation by filtration.
Unlike prokaryotic hosts, fungi have the ability to perform post-translational modifications such as glycosylation and phosphorylation. Modifications like these are required to activate some proteins, including those of human origin. Fungi have more of the cellular machinery to achieve these modifications as they are more closely related to mammals than prokaryotes are. Because of this, fungi from a range of genera have been used to produce recombinant proteins, proteins coded for by DNA that originates from multiple sources. For example, a human gene such as insulin or immunoglobulin G would be inserted into the fungal chromosome to be expressed.
Although filamentous fungi have machinery for some post-translational modifications, they cannot achieve the same modifications as a mammalian cell- addition of a terminal sialic acid for example. Levels of glycosylation differ too: over-glycosylation is a problem, but so is a backlog of protein waiting to be glycosylated. Folding backlogs can also be deleterious, but this can be avoided by overexpression of foldases and chaperone proteins. Protein degradation is another issue, although this can be resolved via the use of mutant strains deficient in proteases related to the protein product.
Optimisation of the growth medium and the insertion of multiple target gene copies downstream of strong promoters are other ways to improve protein production. Another method is the attachment of the target protein to the homologous protein which will enhance the stability and secretion efficiency of the protein.
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