Strange size of pET-22b expression product

Wolfgang Schechinger hubahopp at gmx.de
Wed Feb 25 05:08:51 EST 2004


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:
>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?
>
>--
>\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
>   Mikael Niku             URL: www.helsinki.fi/~mniku/
>   University of Helsinki  Dept. Basic Veterinary Sciences
>       - Mitäkö mieltä olen länsimaisesta sivistyksestä?
>         Minusta se olisi erinomainen ajatus!
>                                              - Gandhi
>////////////////////////////////////////////////////////////
>
>
>
>
Wolfgang Schechinger


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