There's some protease in the yeast fermentation liquor . It cause serious degradation of target protein in the first step of chromatography. Is there some methods to suppress the degradation except lowering the chromatography temperature and adding the protease inhibitors?
Dear Sir. Concerning your issue about how to inhibiting pharmaceutical protein enzymed in the process of the chromatography except lowering the temperature. Proteins contain ionizable groups and the degree of ionization will affect their retention in Reversed Phase Chromatography (RPC). At low pH conditions, suppression of carboxyl group ionization occurs and the amino groups are essentially fully protonated. Thus, the solute can be considered as a single, averaged ionized species. The isoelectric points of most polypeptides and proteins are above these low pH values. For reasons not yet fully clarified, higher selectivity can be obtained when running below the pI value of a polypeptide or protein in reversed-phase systems. Moreover, low ionic strengths can be used at a low pH value, which facilitates recovery and usually gives a better peak shape and more reproducible retention. The following below links may help you in your analysis:
http://www.ispybio.com/search/protocols/purification%20protocol12.pdf
http://www.protein.iastate.edu/docs/542B_lectures.pdf
Thanks
Also you can read the following articles
http://arrow.dit.ie/cgi/viewcontent.cgi?article=1004&context=ehsiart
http://www.ncbi.nlm.nih.gov/pubmed/20978960
https://books.google.co.in/books?id=zTiRJHpKIQoC&pg=PA169&lpg=PA169&dq=avoiding+proteolysis+during+protein+chromatography&source=bl&ots=Y08V0BMshL&sig=Zi3KTU_pKI4kwOn56_zMoEfsJzo&hl=en&sa=X&ved=0ahUKEwiJg5iZzYTOAhVFqo8KHZYeA4wQ6AEITDAI#v=onepage&q=avoiding%20proteolysis%20during%20protein%20chromatography&f=false
https://books.google.co.in/books?id=wiXSCgAAQBAJ&pg=PA593&lpg=PA593&dq=avoiding+proteolysis+during+protein+chromatography&source=bl&ots=kqChfEbaHA&sig=_ZeXh1ksCcDxK6gTrLjRLbmvoNQ&hl=en&sa=X&ved=0ahUKEwiJg5iZzYTOAhVFqo8KHZYeA4wQ6AEITjAJ#v=onepage&q=avoiding%20proteolysis%20during%20protein%20chromatography&f=false
I would go for lowering the pH below your enzyme pI then lower the conductivity to about 1-3 mS/cm than purify you protein on a cation exchange resin. Hopefully your protein will elute away from proteases.
Good Luck
Thank you very much. there is big obstacle in dilute samples conductance from 26mS/cm to 1-3mS/cm because huge volume( about1500L) of yeast fermentation can not be dilute .I use the anion exchange resin POROS® XQ as the capture step .but there is another problem pigment which combining on the resin is difficult to be cleared
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