Hydrophobic protein purification troubles

Hiranya Roychowdhury hroychow at NMSU.EDU
Thu Jul 6 16:03:47 EST 1995


On 6 Jul 1995, Tina Guina wrote:

> I need help with a bit bizzare (?) problem.
> I am trying to overexpress and purify 
> a small, mostly hydrophobic, most likely 
> a membrane protein.
> Despite correct cloning into pET vector, 
> I cannot see the overexpressed protein band
> after running the total (boiled in SDS) cell

If it is hydrophobic and membrane associated, may be you are losing it 
with the bacterial pellet. Another possibility is aberrant mobility due 
to its hydrophobic nature: hydrophobic proteins will tend to bind SDS at 
a higher ratio compared to hydrophilic ones, thereby migrating slightly 
faster on a SDS-PAGE. Thus, you may be expecting a 40kDa band while the 
polypetide is at 35kDa (just an example)

> protein after SDS-PAGE.
> More troublesome is that the fusion of the 
> same protein to the maltose-binding protein 
> (MBP) is sticking to the walls of the plastic
> eppendorf tubes after HPLC purification.

More pure a hydrophobic protein the more will be its propencity to bind 
to plastic (hydrophobic) surfaces. Try siliconizing.

 Does anyone know what I should do to avoid this, > either to prevent
binding to the tubes walls, or > some way to solubilize it without major
damage to > the protein structure? > > Thanks a lot in advance!
> 
> 
> 


			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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