Overexpression studies

Shun'ichi Kuroda skuroda at inherit.biosig.kobe-u.ac.jp
Tue Apr 4 18:53:47 EST 1995


Hi,

>                                            Overexpression studies
>                                            12:12 PM            4/4/95
> I would like to get some input from the yeast community on overexpressing
> proteins in S. cerevisiae. In general (I know it is strongly protein
dependent),
> how much protein can be produced by overexpressing a non-yeast gene from
either
> the Gal1- or ADH-promoter. 

It's not a case by either Gal1 or ADH promoter.  In 1992, I suceeded in
synthesizing 
hepatitis B virus envelope  L protein particles in S. cerevisiae by TDH3
promoter,
of which the level is up to 42% of total soluble protein (see JBC 267 1953
(1994)).
I think this is the highest expression level by S cerevisiae system.

>How did you determine the amount of your expressed
> protein? What is the minimal amount one can detect on Western blots? Does
> anybody know of any references that address this question? I very much
> appreciate all the input and I promise an extensive summary of all the data I
> can collect from you.

First I determine the amount of total proteins by Cu-Folin method.
Second I determine the amount of HBV env L protein particles by densitometric
method by using the purified particles as a standard.

-- 
SHUN'ICHI KURODA
BIOSIGNAL RES CENTER
KOBE UNIVERSITY
NADA, KOBE, 657 JAPAN
E-mail: skuroda at inherit.biosig.kobe-u.ac.jp
URL:http://inherit.biosig.kobe-u.ac.jp/biosighomeE.html
Tel +81-78-803-1255   Fax +81-78-803-0994



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