Why is my protein’s N-terminal blocked (and what can I do about it)?

Alphalyse
Aug 31. 2015

N-terminal sequencing is a requirement according to the ICH Q6B Guideline. This includes structural characterization of recombinant proteins for clinical testing, and to demonstrate comparability and consistency between cGMP batches. It is estimated that almost half of all proteins cannot be directly sequenced by Edman degradation – which causes problems because they have a blocked N-terminal residue. Thus, no sequence data will be obtained if the N-terminus is blocked either naturally or as a result of sample processing.

One problem frequently encountered is that the N-terminal residue is modified in such a way that it does not react with the Edman reagent phenyl isothiocyanate. For example, the blocked N-terminal residue may be an N-acetyl amino acid. It could also be a glycosylated amino acid, or a pyrrolidone carboxylate group.

Of these, you encounter proteins with an acetylated amino acid most frequently. Evidence suggest that about 80% of the soluble proteins in mammalian cells have acetylated N-terminal amino acids.

Examples of covalently modification of the N-terminal amino group of a polypeptide :

  • acetylation − C( = O) − CH3 : Eliminates the positive charge on the N-terminal amino group by changing it to an acetyl group
  • formylation − C( = O)H : The N-terminal methionine usually found after translation has an N-terminus blocked with a formyl group.
  • Pyroglutamate:  An N-terminal glutamine can attack itself, forming a cyclic pyroglutamate group.

 

N-terminal block can also occur after isolation during sample manipulation.

These following steps can help prevent this:

  1. Allow the gel to polymerize well. You need to do this since the free acrylamide might alkylate some amino acids.
  2. Use a reducing agent in the electrophoresis running buffer. (Either sodium thioglycolate (0.1 to 1 mM) or 10 mM reduced glutathione).
  3. Avoid addition of acetic acid in your staining buffer.

Learn more about N-terminal Sequencing and Sequencing Blocked N-terminals.

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