A protein synthesized at a bigger size in E. coli

Hirokazu KOBAYASHI hirokazu at FNS1.U-SHIZUOKA-KEN.AC.JP
Sat Oct 14 06:11:19 EST 1995


Dear Netters:

We would appreciate hearing any comments about our below problem .

We expressed a cDNA encoding a protein of plant origin in E. coli under the
control of tac promoter using a system to produce fused products with
maltose-binding protein.  The cDNA clone had been selected by the ability
of its product to bind with a DNA fragment by south-western blotting. 
According to our sequence data of the cDNA, it seems to encode a protein of
approx. 30 kDa.  After expression in E. coli cells, the product was
purified by affinity column with maltose.  The eluant of the column
contained two peptides as analyzed by SDS-PAGE and CBB staining after
denaturing proteins in the presence DTT.  The lower band corresponded to
the migration position of 66-kDa peptide which was the expected size of the
fused protein, but the upper one seemed to be 100 kDa.  Unexpectedly, only
the upper one showed DNA-bind activity.  We sequenced the upper and lower
bands by protein sequencer after transferring them to PVDF membrane.  We
found only one same kind of N-terminal sequence in both the samples, the
amino acid sequence of which was exactly that of maltose-binding protein. 
All the results let us speculate that the bigger protein might have
attached to conjugating molecules to make lipoproteins, phosphoproteins,
and so on.  I have also once heard a story of probably polyribosylation,
but I do not know its details and references.  It is less possible that the
protein is a glycoprotein, because it was made in E. coli and the result of
PAS staining was negative.

Does anybody have idea(s) about the candidates of the possible conjugating
moiety?

We hope e-mail directly to us (hirokazu at u-shizuoka-ken.ac.jp +
isono at nibb.ac.jp) in addition to this mailing list and newsgroup to easily
identify messages responding to us.  If we receive informative replies, we
would later send the summary of them to this mailing list.

Sincerely,

Hiro Kobayashi
Kyoichi Isono


   /    /  _/
  /___ /   /   /_/  /_ /
_/   _/  _/  _/   _/__/     Hirokazu Kobayashi

Laboratory of Plant Cell Technology
School of Food and Nutritional Sciences
UNIVERSITY OF SHIZUOKA
52-1 Yada, Shizuoka 422, JAPAN
e-mail:  hirokazu at u-shizuoka-ken.ac.jp
phone/fax:  +81(Japan)-54-264-5582

Laboratory of Biological Regulation (Adjunct)
NATIONAL INSTITUTE FOR BASIC BIOLOGY
e-mail:  hirokazu at nibb.ac.jp
phone:  +81(Japan)-564-55-7611




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