1) The synthesized protein may be insoluble. Check its solubility.

2) Factors required for proper activity (e.g., coenzymes, metal ions) may be missing. Since PUREfrex^® is a reconstituted system, it contains no small molecules not relating to transcription and translation. Add the factors required for activity.

3) If the protein of interest requires correct disulfide bond formation, synthesize it in the presence of an additive to promote this formation (e.g. GSSG, DsbC). In that case, use PUREfrex^® 2.1 in which reducing agent can be selected, because the efficiency of disulfide formation is affected by reducing agent.