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Partial cleavage of HA (Hemagglutinin) tag?


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#1 ACL

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Posted 20 February 2013 - 04:35 PM

Hello,

I'm expressing a protein with an N-terminal HA tag which I am using for identification purposes.
A quality-control LC-MS suggests that my protein has 2 populations composed of: 1) "full-length HA tag + protein" and 2) "partially cleaved HA tag + protein"
No other cleavage appears to be evident within my protein since the observed MW agrees closely with the theoretical MW.

My question is, has anyone observed partial cleavage of an HA tag before?
If so, what are the common causes.

Ps. Protein is expressed in CHO cells and is secreted into the media.
Expression construct consists of: 'Signal peptide + HA tag + protein'


Thanks.

#2 bob1

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Posted 21 February 2013 - 11:28 AM

Are you sure that it is cleavage and not skipped expression?

#3 ACL

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Posted 26 February 2013 - 04:31 PM

>Are you sure that it is cleavage and not skipped expression?
I am assuming that it is cleavage since the shortened protein sequence does not begin with a Met.
For skipped expression, what should I be looking for at the DNA level? (other than a second ATG codon).

#4 bob1

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Posted 26 February 2013 - 06:43 PM

I was thinking of a second ATG other than that I don't think skipped expression is possible.

#5 snowchild

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Posted 28 March 2013 - 02:36 PM

Which enzyme do you use before MS? If you use Chymotrypsin, your HA tag is highly possible to be cleaved. Check your HA sequence.




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