From: Jerry McCully
Date: 17 March 2012 06:26
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From: Santosh
Hi Jerry:
There are many instances where we miss going through process of sequencing the entire reading frame, as many sequencing cores provides sequences only upto 600-700 bases, I would recommend sequencing a complete reading frame using mid frame primers and then conclude whether the reading frame is the one that you expect. You may also check for post translational modifications and number of cysteines and secondary structure prediction models using ExPASy.
For as E.Coli expressions try playing with these parameters after you gain enough information about your target gene using ExPASy:
1) Check for the rare codon usage in the target gene, make sure if it has rare codons then try using BL-21 DE3 (RIL) BL21(DE3) (RIPL)?
2) If the gene is toxic then you might not see expression, but if this was the case then you wold not see increase in cell density after induction with IPTG, So the question is were you using LysS or LysE bacterial strain? You may also think of another stringent promoter pBAD containing BL21AI strain (Life Sciences) where you will titrate Arabinose along with 1mM IPTG
3) If you think your target has propensity to form disulphide bonds then consider using AD494, Origami double mutants (Novagen) or SHuffle T7 by (NEB)
4) If you know the binding partner of this gene target then co-expression of this target with a binding partner or associated protein may also yield a better expression.
5) There were case when we used C41(DE3) and C43(DE3) ( By Lucigen) for expressing toxic and membrane proteins.
If nothing helps then to shuttle the gene into yeast (Pichia pastoris), insects or mammalian expression system. I hope it helps and wish you all the best.
Have a great weekend.
Santosh Hodawadekar, PhD
http://www.linkedin.com/in/shodawadekar/
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From: Raji Edayathumangalam
In one case, I had a mutation in the ribosome binding site sequence. If you haven't already checked, it might be worth checking whether the 5'- and 3'- junctions close adjacent to the ORF are all correct to make sure the transcriptional and translational elements do not somehow contain mutations.
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From: Yuri Pompeu
Hello,
Probably a stupid comment on my part, but anyways, make sure your strain has a T7 polymerase! (I have forgotten before).
And I agree with the last idea that sometimes it is worth to just start from scratch. New construct new vector. It may simply just work.
HTH,
Yuri
Date: 17 March 2012 06:26
Dear ALL;
Recently, one of my colleagues cloned a gene (200aa) into pET30a vectors with either a N-ter or C-ter His6 tag. The correct reading frame was confirmed by sequencing.
However, it is weird that there was no protein expression either in the soluble fraction or as inclusion bodies.
Could anyone give some instruction?
Thanks a lot and have a nice weekend,
Jerry
Recently, one of my colleagues cloned a gene (200aa) into pET30a vectors with either a N-ter or C-ter His6 tag. The correct reading frame was confirmed by sequencing.
However, it is weird that there was no protein expression either in the soluble fraction or as inclusion bodies.
Could anyone give some instruction?
Thanks a lot and have a nice weekend,
Jerry
----------
From: Santosh
Hi Jerry:
There are many instances where we miss going through process of sequencing the entire reading frame, as many sequencing cores provides sequences only upto 600-700 bases, I would recommend sequencing a complete reading frame using mid frame primers and then conclude whether the reading frame is the one that you expect. You may also check for post translational modifications and number of cysteines and secondary structure prediction models using ExPASy.
For as E.Coli expressions try playing with these parameters after you gain enough information about your target gene using ExPASy:
1) Check for the rare codon usage in the target gene, make sure if it has rare codons then try using BL-21 DE3 (RIL) BL21(DE3) (RIPL)?
2) If the gene is toxic then you might not see expression, but if this was the case then you wold not see increase in cell density after induction with IPTG, So the question is were you using LysS or LysE bacterial strain? You may also think of another stringent promoter pBAD containing BL21AI strain (Life Sciences) where you will titrate Arabinose along with 1mM IPTG
3) If you think your target has propensity to form disulphide bonds then consider using AD494, Origami double mutants (Novagen) or SHuffle T7 by (NEB)
4) If you know the binding partner of this gene target then co-expression of this target with a binding partner or associated protein may also yield a better expression.
5) There were case when we used C41(DE3) and C43(DE3) ( By Lucigen) for expressing toxic and membrane proteins.
If nothing helps then to shuttle the gene into yeast (Pichia pastoris), insects or mammalian expression system. I hope it helps and wish you all the best.
Have a great weekend.
Santosh Hodawadekar, PhD
http://www.linkedin.com/in/shodawadekar/
----------
From: Raji Edayathumangalam
Hi Gerry,
On occasion, figuring out what is wrong with the current construct might send one on a endless wild goose chase. So if a bunch of ideas with the current construct fail and you still want to try and express in the pET system before moving on to another system, I recommend the pET Expresso system. No need to ligate etc. Add clean PCR product + vector + cells and you directly screen for clones that are made by homologous recombination in bacteria.
--
Raji Edayathumangalam
Cheers,
Raji
--
Raji Edayathumangalam
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From: Yuri Pompeu
Hello,
Probably a stupid comment on my part, but anyways, make sure your strain has a T7 polymerase! (I have forgotten before).
And I agree with the last idea that sometimes it is worth to just start from scratch. New construct new vector. It may simply just work.
HTH,
Yuri
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