4 replies to this topic

[ACL]

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.

[bob1]

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

[ACL]

>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).

[bob1]

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

[snowchild]

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