Strange size of pET-22b expression product

dan su dan_su at hotmail.com
Wed Feb 25 11:09:39 EST 2004


First, let's assume you are talking about reduced SDS-PAGE, excluding the 
possibility of a dimer protein.
Are you sure it is 8 kd, did you included the C-terminal His-tag sequence, 
normally it could be up to 3~4 kd. Also the periplasmic signal sequence, it 
is normally 3 kd, so if you included both the sequence your expressed 
protein is about 15 kd.


>From: "Mikael Niku" <mikael.niku at nospam.vetmed.fi>
>To: methods at hgmp.mrc.ac.uk, proteins at hgmp.mrc.ac.uk
>Subject: Strange size of pET-22b expression product
>Date: Mon, 23 Feb 2004 14:43:58 +0200
>
>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
>////////////////////////////////////////////////////////////
>
>
>

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