I have seen that the mass of Bacillus subtilisin is about 27.5Kda.Light
chain is about 20Kda,while heavy 55Kda.
The largest molecular weight is about that of intact IgG.I will not try to
get any further,but multiple sequences could be due to the two IgG
chain,and you recognized that one sequence is similar to Kappa chains.
Did you do the sequencing on elute bands from the gel?I would expect that
you have IgG still present,and you were
looking at that protein.As for your subtilisin,did you get significant
activity.
Now,I will go to a few of our experiments with expressed proteins.We have
never found a significant HPLC peak with
the expected sequence,nor other evidence that we,conclusively, had what we
thought we had.There was only one exception when we expressed a protein in
a bacterial system,and we got a ton of material.
I hope that you find what you are looking for.In my case,all the lack of
success make me somewhat cynical.
"Linda Siconolfi-Baez" <lsiconol@aecom.yu.edu>@aecom.yu.edu> on 11/08/2000
06:38:44 PM
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Subject: ProtPrep/ProtSeq
Can any one offer possible suggestions for the following puzzle?
-To confirm the identity of Subtilisins from an unusual eukaryotic
species, Edman
sequencing was used .
-The starting material was from two sources, either the eukaryotic cells or
a
Bacculovirus system expressing Subtilisin.
-Immunoprecipitation (using two different polyclonal rabbit Antibodies
coupled to
Protein A but following the same protocol) was used to isolate the
Subtilisins,
followed by SDS/PAGE and blotting.
-Four runs from four bands ranging in size from 85 to 140 kDa generated
multiple
residues in each cycle.
-Subtilisin sequences were not present.
-All four sequences appear to be the same sample. (Relative yields of all
the amino
acids in the respective cycles in all the runs are comparable, despite the
antibody
used and the protein size.)
-Homology searches pulled out N-terminii of several Ig Kappa chains.
Linda
Big Apple Minute: Asking for the Giants to meet the Jets in the Superbowl
does seem a
bit stingy after the Subway Series, but we are the BIG apple after all.
P.S. I would like to take more time to think through the membership's
response to
the cancellation of the PARG study before re-addressing the group on the
issue.
Linda Siconolfi-Baez
Laboratory for Macromolecular Analysis
Albert Einstein College of Medicine
Ullman 405
1300 Morris Park Avenue
Bronx, N.Y.10461
718-430-2864
lsiconol@aecom.yu.edu
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