I want to remove hemoglobin from aspergillus cell culture supernatant. It has been used as a sole nitrogen source in order to make aspergillus produce proteases. I want to remove it in order to identify proteases using LCMS (which is not possible in the presence of hemoglobin).
How much haemoglobin is present in the medium and what source did you use (eg commercial preparation)?
Hi Bassel, I don't know of any quick and easy procedure to remove hemoglobin from a sample that will not bind any other proteins, besides using immobilized anti Hb antibodies or immobilized haptoglobin
(If anybody knows of an easier procedure, I will be very interested in it).
Probably a better strategy is to isolate your proteinases from the Hb then the other way around. There are many affinity resins for proteinases available.
Hi John, i used 5 mg/ml (Hemoglobin from bovine blood, Sigma)
Hello Steingrimur,
i found a kit for hemoglobin deleption, however i am not sure that it will not remove my proteases!!
could you provide me with more information about affinity resins for proteinases please
If hemoglobin was used as a carbon source, and as an inducer for proteases, it means that after cultivation most of it will be degradated and metabolized by Aspergillus.
To separate hemoglobin fragments from your proteases you can use a gel filtration method, or ultrafiltration using a 10,000 da cut of membrane.
HNS
Hector, hemoglocin is used as nitrogen source. i performed gel filtration in order to remove the peptides of degraded hemoglobin. however i had discovered that it was not completely degraded by aspergillus proteases which affected the LCMS results later. i can try to reduce the used amount of hemoglobin but i am not sure if it would be completely degraded by aspergillus proteases.
Hi Bassel, there are a lot of resins available commercially that will isolate specific proteinases, but you will have to give more information about the class of proteinases you want to analyze.
Are they serine, cysteine, aspartic or metalloproteinases?
It would also help if you know what peptide bond(s) they cleave.
Hello Steingrimur, they are mainly serine and metallo proteases (Aspergillus produces up to 136 proteases but most of them are uncharacterized)
Hi Bassel, that is a lot of proteinases! I hope you are only focusing on some of them.
Anyway, I have used affinity resins to isolate serine proteinases. For trypsin-like serine proteinases that cleave after Arg or Lys, you can use immobilized benzamidine. For chymotrypsin-like serine proteinases that cleave after large hydrophobic aa, like Tyr, Trp, Leu, Phe, you can use immobilized 4-phenylbutylamine.
Affinity purification of metalloproteinases is more tricky since their active sites (mostly) do not form a covalent intermediate with their substrates.
However, I did find some papers that use iminodiacetic acid-Sepharose to bind the coordinated active site Zn of metalloproteinases. Personally, I haven't used this resin, but these papers look convincing.
http://scholar.google.com/scholar?q=%22Iminodiacetic+acid+Sepharose%22+metalloproteinase&hl=en&as_sdt=0&as_vis=1&oi=scholart&sa=X&ei=VAxAVKOtF5GlyQTDkoLIDA&ved=0CB0QgQMwAA
thank you so much for your detailed answer Steingrimur,
i want to identify as much as possible of secreted proteases, so i am trying different approches
Hi Bassel, you are welcome.
Also Sigma has a bunch of affinity resins that you can choose from:
http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=9599892
http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=9599506
http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=9599960
http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=9599505
According to this paper, Aspergillus proteinases prefer to cleave hydrophobic aa bonds:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117871/
Sigma has some immobilized hydrophobic aa.
Good luck!
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