pET expression system
bllim at hkucc.hku.hk
bllim at hkucc.hku.hk
Thu Mar 2 21:39:28 EST 1995
In article <S.D.Minchin.138.000A9E6A at bham.ac.uk>, S.D.Minchin at bham.ac.uk (Steve Minchin) writes:
> In article <3ijklg$900 at forged.passport.ca> scontz at passport.ca (Marc Visconti) writes:>
>>I'd like to thank everyone for being so kind as to send me their 2 cents
>>worth regarding my problem. However, I realize I may not have fully
>>explained my problem. The term "increadible induction" was in reference to
>>my Western blots. With the other expression systems used, I never saw a
>>clear cut difference between my induced and non-induced fractions. I made
>>a conclusion that my protein is soluble by running soluble and insoluble
>>lysates after sonication. Virtually all of my product, when detected with
>>the S-protein Western kit, was soluble.
>>Currently my protocol requires plating transformed cells on kanamycin
>>plates (50 ug/ml), picking a colony, growing o/n to an OD-600 of 1.5- 2.0.
>>This is used as innoculate into fresh media (LB for now) with 50 ug/ml
>>kanamycin. These are grown to OD-600 0.4-0.6, and then induced with 1mM
>>IPTG for 2-3 hours. These are then spun down, and resuspend in Sample
>>running buffer with SDS, boiled and run on SDS-PAGE. Am I doing something
>>wrong or missing something? Thank you in advance for all of your help.
>>scontz at passport.ca
>>marcvis at resunix.ri.sickkids.on.ca
> We had a similar problem when we tried to overexpress the human cardiac TnI in
> E. coli using the pET system. We first looked at codon preference with no
> success. So we made successive deletions from the N-terminal coding region,
> this showed us that lack of expression was due to the first five codons. We
> developed a screen for expression and then mutated the first five codons
> (maintaining the coding potential). We picked up mutant which over expressed
> TnI, this contained two changes, which were both in wobble bases and therefore
> the mutant gene produced the same peptide sequence as the wild-type gene.
> If you want more information we have published the work.
> Al-Hillawi, E., Minchin, S.D., and Trayer, I.P.
> Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli
> and there purification and characterisation.
> Eur.J.Biochem. 225:1195-1201, 1994.
> All the best
> Steve Minchin
I am trying to use the pET vector obtained from Novagen for protein
Now I have subclone the fragment in pET and would like to sequence the
insert. What primers should I use for sequencing?
The T7 promotor primer and the T7 terminator primer?
Could anyone give me the sequence of these two primers or the primers
that they use for sequencing? It takes time for ordering and I would like to
synthesis the primers my self.
Thanks in advance!!
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