A protein synthesized at a bigger size in E. coli?

Hirokazu KOBAYASHI hirokazu at FNS1.U-SHIZUOKA-KEN.AC.JP
Thu Oct 19 02:33:32 EST 1995


Dear Netters:

We would appreciate hearing any comments about our below problem.

We have once posted the similar messages to
methods-and-reagents at net.bio.net, but we have received no replies. 
Therefore, we are cross-posting this to proteins at net.bio.net and
arabidopsis at net.bio.net, since we thought that
methods-and-reagents at net.bio.net was not suited to expecting answers to our
problem.

We expressed a cDNA encoding a protein of plant (Arabidopsis thaliana)
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 (therefore,
disulfide bonds if any are expected to be cleaved).  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-binding 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 been associated with conjugating molecules to make
lipoproteins, phosphoproteins, poly-ADP ribosylated ones, and so on.  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 similar experiences or any ideas about the candidates of
the possible conjugating moiety?

If we receive informative replies, we would later send the summary of them
to these mailing lists.

Sincerely,

Kyoichi Isono
Laboratory of Biological Regulation
NATIONAL INSTITUTE FOR BASIC BIOLOGY
e-mail:  isono at nibb.ac.jp

Hirokazu Kobayashi
Laboratory of Plant Cell Technology
School of Food and Nutritional Sciences
UNIVERSITY OF SHIZUOKA
e-mail:  hirokazu at u-shizuoka-ken.ac.jp
Laboratory of Biological Regulation (Adjunct)
NATIONAL INSTITUTE FOR BASIC BIOLOGY
e-mail:  hirokazu at nibb.ac.jp




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