Glycoprotein

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Fri Apr 6 10:17:07 EST 2007


Dairpoosh wrote:
   
>   Im looking for a method for isolation of Glycoproteins
>   from plant cells.I would be very grateful if someone can provide
>   me with a method and a refrence.

There is no such thing as *the* method to purify proteins, each protein
is different and needs its own combination of methods. 

Now since you are talking about glycoproteins I presume that you are
dealing with a transmembrane protein. To isolate those, you homogenise
your cells and isolate the compartment where your protein lives (ER,
Golgi, PM, lysosome...). There are standard methods for that, check e.g.
Meth. Enzymol.

Then you solubilise the membranes with a suitable detergent. There are
hundreds of detergents out there, dozens are in common use for membrane
protein isolation and may be one or two will solubilise your particular
protein without destroying its activity. You have to find out which
ones. The following is still the first paper to read, despite being 30
years old:

@article{Hel-75,
        AUTHOR= {A. Helenius and K. Simons},
        TITLE= {Solubilization of membranes by detergents},
        JOURNAL= {Biochim. Biophys. Acta},
        VOLUME= {415},
        YEAR= {1975},
        PAGES= {29-79},
        LANGUAGE= {engl}
}

The resulting protein/detergent mixed micells can be purified by
conventional chromatographic methods, except that the detergent needs to
be present at its cmc at all times, to prevent protein aggregation. Note
that cmc depends on buffer composition! Measuring the cmc is quite easy:

@ARTICLE{Cha-84,
         AUTHOR= {A. Chattopadhyay and E. London},
         TITLE=  {Fluorimetric determination of critical micelle
concentration avoiding interference from detergent charge},
         JOURNAL={Anal. Biochem.},
         YEAR= {1984},
         VOLUME= {139},
         PAGES= {408-412},
        LANGUAGE= {engl}
}

You can use the sugar groups on your protein for purification with a
lectin column.

Once you have a pure protein, you need to get it out of the detergent
micells into a phospholipid membrane again. This "reconstitution" step
is the blackest of all arts in biochemistry, but recently some
theoretical basics for the process have been worked out, for a review
see:

@article{Rig-98,
        AUTHOR= {J.L. Rigaud},
        TITLE= {Membrane proteins: {F}unctional and structural studies
using reconstituted proteoliposomes and 2-{D} crystals},
        YEAR= {2002},
        JOURNAL= {Braz. J. Med. Biol. Res.},
        PAGES= {753-766},
        VOLUME= {35},
        LANGUAGE= {engl}
}

All in all, this is a program for a nice PhD thesis. Good luck!


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