HELP! Problems with the expression of HSP 70.

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Sun Nov 20 09:27:08 EST 1994


In article <1994Nov18.093935.33203 at cc.usu.edu>, sl16s at cc.usu.edu wrote:

> I am trying to express a fungal HSP70. I have tried pUR278 vector first.  I
> cloned my insert in the three different reading frames. but did not see any
> expression. I changed the vector to pET 21 and subcloned into the three
> different RFs again. but still not able to see any expression.  I know the
> first 300 and last 300 base pairs of the insert sequence. There is a start
> codon and a poly A tail.

  I am not familiar with pUR278 but pET21 seems to be a good choice.  The
T7 lac combination should effectively block the expression of Hsp70 until
induced.
  To be able to figure out what is wrong with your clones we need a bit
more information.  The pET21a-d vectors have a Ribosome binding site
downstream of the promoter and a Met initiation codon at apropriate
distance.  These vectors add a T7-Tag leader peptide to your protein.  You
mention that there is a start codon in your insert and that you have cloned
into pET21 in all three reading frames.  This does not seem very logical. 
If you know the sequence at the start of your insert you should simply
choose a vector that places the ORF of your insert in frame with the leader
tag.  Take care that there are no stop codons in front of the ORF or you
will only be expressing the T7 tag.
  If you are using the pET21(+) vector to express Hsp70 without a leader
tag, you must be aware that the plasmid does not provide a ribosomal
binding site in front of the polylinker.  Since your insert is a eukaryotic
cDNA the chances that it will have a functional E. coli SD sequence at the
right distance from  the initiator Met are indeed very small.  
  Another important thing regarding protein expression using the pET series
is the strain used for expression.  Of course the strain must have the gene
for T7 RNA polymerase, but that is not always enough.  Somtimes it is
worthwile to try a few different strains.  The popular BL21 strain in
particular seems to refuse to express some proteins without any obvious
reason.

Good luck !

Zophonias



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