Strange size of pET-22b expression product

Hello!

I'm expressing three fragments of a mammalian membrane protein in E. coli,
using the pET-22b expression vector (which contains a periplasmic secretion
signal and a C-terminal His-tag).

The two extracellular fragments are OK but the cytoplasmic fragment appears
about double the expected size (about 15 kD, should be about 8 kD) in
SDS-PAGE. The plasmid sequence is fine, the protein is strongly expressed
and succesfully secreted, and is succesfully purified with Ni-NTA.

Any ideas what could cause the strange size in SDS-PAGE? Is it practically
possible that all the terminators are somehow ignored and a longer sequence
gets transcribed & translated? I guess the bugs shouldn't be able to do any
significant modifications which would increase the molecular size?

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Mikael Niku URL: [Only registered users see links. ]
University of Helsinki Dept. Basic Veterinary Sciences
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Dear Mikael,

cytoplasmic - very polar or even highly charged? - might attract more SDS -
*apparent MW* looks heavier then reality? Does the protein sequence give
you some clues?

Just some thoughts,

Wo


At 14:43 23.02.2004 +0200, Mikael Niku wrote:
Wolfgang Schechinger


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