generally bacteria is used for protein expression in purification whenever protein is required in large quantities for structural studies. Is it possible to get such large amounts of proteins from mammalian cell lines?if the protein is tagged is purification possible? can anyone suggest the protocol for this purpose?
It is possible to make large amounts of protein from mammalian cell lines, and this is often done in the biotechnology industry. Gram/liter of culture yield is possible for antibodies, for example. No doubt this requires a fair amount of optimization.
Yes, it is possible to produce huge amounts of specific proteins from mammalian cell lines.
Yes, there are large scale protein production is possible in different mammalian cell lines. Specific eukaryotic expression vectors are used to clone the desired gene and further for protein expression.
I have read that vectors containing strong promotors with (his)6 tags are used for some proteins. i wanted to use the protein for structural studies so I wanted to modify the vector containing the gene of interest. the protein I want to purify has the molecular wt of 85Kda and is a membrane protein. will I get sufficient protein for my study?
It is much more difficult to make large amounts of integral membrane proteins, and more difficult to crystallize them. For some proteins that have a large cytoplasmic or extracellular domain connected to a transmembrane domain, it is possible to make a construct of the soluble domain only and express it in large amounts.
Some membrane proteins are peripheral, rather than integral, and may be expressed in soluble form by deleting only the small part that attaches to the membrane.
Dear Sansrity,
The over expression of proteins in mammalian cells is possible, but it is considerably more difficult, labour intensive and expensive. There are two ways of doing it: transiently or stably. You generally get better yield via transient transfection, but over time the cells will lose the plasmid and stop expressing it. Additionally, it requires the use of viruses to introduce the vector. Stable transfection means that your cells retain the plasmid over time. However, the yield of protein may be lower.
For a detailed guide I would direct you to this publication on Nature Protocols: http://www.ncbi.nlm.nih.gov/pubmed/22322218
I would also point out that your protein being a membrane protein adds considerable difficultly in an already challenging system. They are generally expressed at low copy numbers per cell, and may potentially be toxic to the cells.
I hope this is helpful to you.
Good luck,
James
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