Periplasmic extract/His tagged Prot??

Mxm pemanuel at umabnet.ab.edu
Fri Mar 24 10:46:11 EST 1995


Netters:
  I am expressing a His 6 tagged Fab antibody fragment generated by phage
display in XL-1 Blue cells. The clones were isolated in SurfZAP and the
excised pSurfscript clones have been altered to insert 6 histidines rather
than the cpIII gene. I have a few questions........

1.  I induce with IPTG and the pel B is supposed to excrete Fab into the
media-Will I lose a significant proportion if I toss the media and go
straight for the periplasmic portion?  I have heard that most of it is
trapped there anyway.

2. I need a good protocol for making periplasmic extracts.  I want to pass
this extract over Qiagen Ni+-NTA resin to bind my Fab.  I am seeking a
detailed protocol paper (Not a PNAS or shortened methods paper!) which
describes how to make a periplasmic extract which may be compatible with
the Ni-NTA columns.

3.  Does anyone have experience with native purification on nickel
columns.  What advice can you offer?

4.  Does anyone have experience with expression of antibodies in
bacteria?  What are the yields?  Do you express at room tempt for days or
what???

Thanks in advance for any help. 
    Peter



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