Protein expression

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DinaLida
DinaLida's picture
Protein expression

To produce an antiserum, I am going to express the coat protein (CP) of a plant virus in E. coli using the pMAL protein fusion and purification system (New England Biolabs). When I insert the CP fragment in the expression vector, the protein will have nine additional amino acids in the amino end. How could I know if these additional amino acids may affect the protein expression or the antiserum production? What should I do in this case?
Thank you in advance.
Dina

Richard Taylor
Richard Taylor's picture
I would recommend breaking

I would recommend breaking this question down.

1. How do you measure the expression levels of any protein?
Apply your answer to this situation.

2.How you test the activity of an antibody / antiserum
What's the simplest way

You are creating a polyclonal antibody - what consequences does this have?

If you did get antibodies to an epitope containing the nine additional amino acids produced will this matter - will it affect the results of your activity test?

Jason King
Jason King's picture
I had a look at NEB's website

I had a look at NEB's website and the pMAL system they describe appears to have more than 9AA at the N-terminus. From the cartoon it looks like at least one domain of the Maltose Binding Protein (MBP).

Following protein production in the Colis, the MBP domain is cleaved off to leave only your coat protein. From the cartoon it appears that there is nothing extra at the N-terminus.
"This allows the protein of interest to be cleaved from MBP after purification, without adding any vector-derived residues to the protein"

see: http://www.neb.com/nebecomm/products/productE8000.asp