Bacterial protein expression and purification discussion
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Bacterial protein expression and purification discussion
I am working with the pBAD bacterial his tag expression system from
Invitrogen, and am trying to purify the his-tag protein. I have checked
a number of methods to lyse the cells to release the protein, but in
each case the vast majority of my protein stays in the pellet. I have
used lysosyme, freeze thaw, sonication and NP-40 (NP-40 followed by
freeze thaw seemed to work the best). I even tried 8M urea, even though
I'd prefer to use native conditions. Does any one have any
recommendations of how to generate a lysate which will contain the bulk
of my protein?
Do you know that your recombinant protein is not going into inclusion
bodies? They are quite resistant to most lysis methods and will require
harsh treatment to solubilize - on the other hand, the inclusion bodies
can be used as a first round purification step.
How would one tell if the protein expressed is going into inclusion bodies?
>
If your protein is in the pellet after cell lysis and
centrifugation (if that is your question). If what you want
to know beforehand if your protein is likely to go into
inclusion body, this is more difficult. The presence of
disulphide bonds is likely to result in protein going into
inclusion body. The presence of hydrophobic patches may
also affect solubility, but that is difficult to tell
without knowing the structure of the protein, although
structure prediction can sometimes help.
> A separate question: I would like to express a recombinant protein in
> E.coli, ideally getting high yields in the supernatant (I can add a signal
> peptide in front of my protein). Is anyone aware of any good
> protocols/vectors for this job?
Generally, the presence of a fusion partner helps with
protein solubility, although lots of proteins are soluble
without any fusion partner. If you are worried about
solubility, maltose-binding protein (use pMal vectors from
NEB) is supposed to be best at promoting solubility. See
Kapust RB, Waugh DS, Protein Sci 1999 Aug;8(8):1668-74
Expressing your protein at low temperature can also help
with solubility. There is, however, no guarantee that any
protein will be soluble. You can always try to refold your
protein should it ends up in the inclusion body.
We have had good luck growing cells at lower temperatures than 37oC (as low
as 15oC). The slower metabolism seems to promote correct folding of your
protein.
Last update 18-Jun-2002, Rating Good of 14 votes.
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Hello,
I purified a 35KDa His-Tag protein in 250mM IMIdazole in 0.3M NaCl and Na Po4. Afte freezing and thawing it became turbid
what should I do\?
Thanks
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hello sir,
I am working with GST TAGGED inclusion body protein .Give me the suggesions for solubulization and refolding of the fusion protein.
thank you sir
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In our lab we express recombinant human interleukin 2 with his tag and now i want to know the procedure of its purification
is there anyone can help me?
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In our lab we express recombinant human interleukin 2 with his tag and now i want to know how i can purify it.I saw your message now and i appreciate you if send me the procedure of your work for purification
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Dear Friends,
I wanna find a nucleotide sequence of a protein with interest function, but neither DNA seq nor AA seq is available, first how can i extract the exact protein? i will attempt to Sequence 7-8 amino acid from both N-terminal and C-terminal, then with reverse translation attain the whole DNA Seqence,
if you have any idea please assist me,
thanx
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I am using pTH11 plasmid to express my protein in DK8 strains of E. coli. I get good growth of DK8 strain but I am having problems. Inspite of getting high cell yield, I am getting less concentration of protein i.e. 4 mg protein from 41 gms of cells, obtained from 4 litres of culture.
I have been working on this since couple of months ago, I changed different parameters but the problem still exists. If someone can give their ideas, it would be great.
thanks
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1. i am working with pET28a vector. expression is very good, but protein going into inclusion bodies. I dissolved it into 6M Urea but with desired protein lots of contaminated protein comeing. When i dissolved pellet into 6M Gu.HCl, then protein was comeing into flow through, no binding. I am using HisResin. Can somebody help to solve this problem?
2.I have tried to get protein into supernatant but fails. I tried with low temp.(20-30C, high NaCl conc. upto 1.5 M, Triton-X-100, different buffer like sodium phosphates, Triss, Sodium dihydogen phosphates with pH(5.5 to 8.8).
Please me what to do?
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I have a question regarding His tagged protein purification.Whenever I give lysozome treatment to dissolved pellet ,after the completion of Induction and before the sonication, the pellet becomes very slimy and viscous.Even after centrifugation, the supernatant is not cleared and is very viscous.But I am doing same thing for GST-tagged protein purification, I never faced such problem.
Can anyone suggest me some thing to solve this problem?
I will really looking for the answer
Thanks.
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I have expressed my gene in pMAL-c2X expression vector. I am getting 84 kDa protein band on PAGE gel, but on SDS-PAGE gel it is cleaving in two band (43 kDa ;MBP and 45kDa band). Please find the solution why is is happening.
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I try to produce antiCD45 BC8 scFvSA fusion protein in XL1-blue cells. With amp selection, I got a terrific result, but switched to kan as selection drug on the plasmid, the result is terrible. However, a kan selection works for another fusion protein e.g. CD20 scFvSA although it is not as good as amp one. My BC8/kan strain just poorly grows (partially lysed) during the fermentation. Anybody knows why. Thanks.
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I am trying to express tetanus toxin fragment C in V.cholerae vaccine candidate strain. Although my reading frame is correct and I get a good result with real time PCR, but I failed to get a band in western blot. My band is expected in supernatant as I am using a promoter with signal peptide and propeptide.
Could you please help me in this regard?
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hello sir,
Iam srikanth now working on the inclusion body proteins ,Iam having a very basic doubt that the Pi (isoelectric point ) of the protein varies between the misfold and correctly folded protein
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hi
my protein is GST-Tag and i try to purify it from pellet.what do you prefer to do.my protein is form inclusion body .
thanks
ali
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hi
i m very new for protein expression. after having the cprrect reading frame and coorect insert in pQE 31 vector of 952bp in not getting the protein, plz help me.I have just strated my protein work.
thanks
atul
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how can I reuse NiNTA matrix (for the same protein purification)? what should or souldn't I do? has anyone some good experience?
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I have to make a choice between going for affinity columns or using magnetic beads to attach the
protein I purified , so as to find its interacting partner( in low amts atleast). Is there any recommendation
that someone likes to forward me?
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I am trying to purify a protein tagged to His. I could purify the protein in small Qiagen collumns but could not purify using packed Ni-NTA columns. The expression of the protein is less .
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I am trying to purify a protein tagged to His. I could purify the protein in small Qiagen collumns but could not purify using packed Ni-NTA columns. The expression of the protein is less .
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Who could tell me the sencondary structure of interleukin-1 receptor antagonist?How can I improve the solubility of inclusion body of IL-1ra.Many thx for ur considering.If U know the knowledge plz contact me at 0086 23 62813515 or thd882000@yahoo.com.
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Can u suggest me some good vector system which will give small his tag(6residues) at N-terminus or c-terminus immediate to the gene?
The vector should have promoter other than phage promoters (i.e they should not have any requirement for specific phage RNA pol) as gene which i want to clone is toxin for e.coli and can be expressed only in resistant stains.
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Many comments on your problem:
First
for solubilizing your protein you can use Guanidium hydrochloride 6M it's the harshest solubilizing agent.
second
Solubility is probably the most difficult issue, lower temperature for induction time and lowering the expression is usually helping for soluble production.
Third
Of course denaturation can be a problem there are some refolding protocol available both after purification or more recently directly on the column by washing the bound protein with a gradient wash of decreasing concentrations of denaturing agent or by using oxidized glutathine in saline buffer. After refolding the protein can then be eluted with imidazole gradient(in the case of His tag)
Hope that might help you. there are of course tons of others possibilities like co-expression of chaperones molecules to help proper folding and therefore solubility but unfortunately it vary from protein to protein and nobody can give real prediction
So jut try out as much as you can.
Feel fre to e-mail if you have specific questions
Pesji
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