Producing the proteins in cell free system or bacterial expression can affect the removal of the start methionine. Although the same rules of a small amino acids next to the start methionine apply, different methionine aminopeptidases tolerate certain small ones better. Depending on the which cell-free kit you use, you may have not removed the methionine, which may be important in protein binding.
Protein N-Terminal Processing: Substrate Specificity of Escherichia coli and Human Methionine Aminopeptidases Biochemistry 2010, 49, 5588–5599 A second important consideration is that in cell-free systems other post-translational modifications can be a hit or miss depending on the cell-free kit and/or type of modification. For example acetylation occurs in transdirect insect line N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry PROTEOMICS Volume 6, Issue 16, No. 16 August 2006, Pages: 4486–4495 but not wheat-embryo Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system Protein Expression and Purification 52 (2007) 59–65 though it can be achieved by the addition of Acetyl-CoA.
