Keywords: Golgi membrane protein; mammlian system; overexpression is troublesome
I'm currently on a project needs tons of a membrane protein on Golgi apparatus for structural analysis. However, due to the small size of Golgi (using HEK293T now) and a issue of the protein that when overexpressed, the protein would mislocalize among the whole cell.
The membrane protein, let's call it protein B, seems to be saturable on golgi apparatus according to a study, overexpression would result in mislocalization and strange modification. While directly purifying endogenou protein B is way too insufficient for structural analysis and....not beauitful(?).
I'm seeking a way to get sufficient amount of protein B, considering using cell lines with bigger Golgi apparatus, and even inducing Golgi Swelling! (sounds crazy tho).
Are there any recommendations or suggestions?
(PS: mammlian expression systems are preferred.)
In the old days, bovine brain was used to purify factors involved in membrane traffic in quantities sufficient for biochemical studies. Another way is liver Golgi membranes from mice. For both, there are protocols published. If you have specific reagents for your "Protein B" (antibody for IPs), then these are viable methods.
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