Hi Angela,

Are you saying that you've used an N-terminal His tag?  Also is your protein secreted? and have you calculated the pI of your protein? I use Protparam (http://web.expasy.org/protparam/) to calculate numerous physical and chemical properties of proteins to help determine a suitable purification strategy.

I have had very mixed success purifying his-tagged proteins in Pichia and generally for secreted proteins I prefer to use ion exchange as a capture step followed typically  by gel filtration.  If you have a suitably basic protein with a pI greater than ~8 then diluting the culture supernatant and applying to a cation exchange resin (at pH 5 ish) often works well.

Another problem that you might encounter is removing non-specifically bound sugars from your sample.

Best of luck

Dear Angela,

I wondered how you know that it has lost the tag, when it lost (loses) the tag in your isolation sequence, and whether there is any reason it should lose the tag (for instance, you have a cleavage site engineering between your protein sequence and the tag, and for some reason you think the site might be cleaved by a pichia protease, etc)?

If you do an anti-his western on the lysate, do you see your protein?  I guess I am wondering whether you know you have expression of your protein in the first place?

Dave

Hi David, I know that my protein was expressed because I measured activity with the specific substrate at  different times of expression *0 - 144 h( and I don´t have activity at or before the induction process, I have activity at 96 h of expression. Currently I´m doing bioreactor cultures and I have my protein active. So, I did western blot wth specific antibodies against my proteins and I could see bands, but when I did WB with anti-his I couldn't see anything. The same occurs with His purification... so I sequenced the first aminoacids of my sample by Edman sequencing to verify the presence of my His sequence, and I could not found it, So that is the reason why I say that my protein doesn't have the his tag sequence.

Angela

Hi Angela,

Are you saying that you've used an N-terminal His tag?  Also is your protein secreted? and have you calculated the pI of your protein? I use Protparam (http://web.expasy.org/protparam/) to calculate numerous physical and chemical properties of proteins to help determine a suitable purification strategy.

I have had very mixed success purifying his-tagged proteins in Pichia and generally for secreted proteins I prefer to use ion exchange as a capture step followed typically  by gel filtration.  If you have a suitably basic protein with a pI greater than ~8 then diluting the culture supernatant and applying to a cation exchange resin (at pH 5 ish) often works well.

Another problem that you might encounter is removing non-specifically bound sugars from your sample.

Best of luck

I would still try a restriction digest of your plasmid if you can just so you can understand what is going on.  Since your enzyme has actidily you could try substrate affinity chromatography for purification?  Best regards, Dave

Hi Andrew, yes my proein is secreted, and it has pI ~6 or lower. I've used anion exchange and gel filtration as you suggest me.  

So How can I remove the non-specifically bound sugars that you say?

Sorry should have said sequence your insert -make sure tag is still there - it's been a long day!  Dave

During maturation or secretion, is the protein cleaved?

Does the 75kd mass spec data show histidine containing proteins?

Does the dimer interact with anything else?

Could it be cleaved by culture medium components?

What does crude extract look like on a coomassie gel?

What does the control culture western look like?

Are the dimer or its partners covalently linked?

As mentioned above, search the web for protein modification and cleavage prediction software and look for the cleavage site.

From http://www.uniprot.org/help/mod_res

9. Flavin-binding

Enzymes involved in electron transport, oxidation and reduction often contain a flavin group (flavin mononucleotide [FMN]) or flavin adenine dinucleotide [FAD]) as a cofactor. When the cofactor is covalently bound to the protein, this is considered as a post-translational modification and is annotated in the ‘Amino acid modifications’ subsection. When the cofactor is not covalently bound, the binding region is annotated in the ‘Nucleotide binding’ subsection. The flavin can be transferred to the hydroxyl group of a serine, threonine or tyrosine, to the one or the other nitrogen of a histidine or to the sulfhydryl group of a cysteine. Flavin-binding has been observed in all organisms.

Examples: P21398, P09788, Q9UI17, Q752Y3, Q9K0M5, Q9KPS2, O34627, Q48303, P40875

for candidates, google these:  "flavoprotein pichia pastoris"  and "covalently flavoprotein pichia pastoris"

http://www.researchgate.net/post/How_to_overcome_the_elution_of_non-specific_bands_in_protein_purification_by_Ni-NTA_with_His-tag

http://link.springer.com/article/10.1007%2Fs00726-003-0015-y

http://www.pnas.org/content/89/10/4544.full.pdf