I am using the Hansenula Polymorpha for the production of rhPTH and I am suffering from the degradation of the PTH starting from 37 hour of fermentation duration and I am losing more than 50% of the yield. Any ideas what to do for this matter? How can I use the proper protease inhibitor cocktail? Is there any other options for inactivating the proteases than the protease inhibitor cocktails?
Are you making the full-length protein of 84 residues or the bioactive N-terminal 1-34 fragment? What I'm thinking is whether it would be possible to continuously separate the PTH from the culture medium containing proteases by an ultrafiltration process, such as tangential flow filtration or diafiltration. If the proteases are much larger proteins than the rhPTH, then a suitable molecular weight cutoff membrane could be used for the purpose. So this would work best with the 1-34 fragment.
Another possibility is to seek out/construct strains of the fungus that do not excrete the proteases or that lack them entirely.
Thank you very much fir your answer.
I am using the bioactive N-terminal 1-34 fragment and i am in the final stages of the development so i wanted to seek manipualting some parameters like pH or temperature or add protease inhibitor to inactivate the proteases but i don't know what could inactivate them.
Hi Peter, do you know the amino acid composition of the bond(s) cleaved?
If it is a proteinase with trypsin-like specificities then you can include high concentrations of free lysine, arginine, benzamidine, etc in the media to serve as competitive inhibitors of the proteinase(s) without being overly toxic to cells.
The same strategy can be used for chymotrypsin-like proteinases. Include tyrosine and/or other non-toxic competitive inhibitors.
Thank you very much Steingrimur for your answer.
I want to add that there is no yeast extract added to the feed solution, only glycerol wheat peptone and methanol. So do you think the absence of the yeast extract in the feed could be the reason behind amino acid limitation? Do you think if I added yeast extract to the feed solution, this could supply the media with enough amino acids and peptides to act as competitive inhibitors for the proteases.
Hi Peter, competitive inhibition of these proteinases by adding amino acids and peptides is the easiest and least toxic way to curb their activities.
You can only find out how effective this is by doing pilot studies.
Good luck!
Hi Steingrimur
Thank you Steingrimur very much for your answer.
I will test adding amino acids or/and yeast extract to the media solution. I want to add that the major protease which is degrading the PTH is the yapsin 1 protease of the aspartic acid protease familay. The name of the protease is HpYps1p, which decompose proteins containing a basic or dibasic amino acid residues. It specifically decompose the PTH-34 into 3 degradation products. What amino acids do you think i should try to test and add so i can competitively inhibit the yapsin 1 action.
Thabk you again for your answer and for your help.
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