Unexpected instabilities explain batch‐to‐batch variability in cell‐free protein expression systems

Cell‐free methods of protein synthesis offer rapid access to expressed proteins. Though the amounts produced are generally only at a small scale, these are sufficient to perform protein‐protein interaction assays and tests of enzymatic activity. As such they are valuable tools for the biochemistry and bioengineering community. However the most complex, eukaryotic cell‐free systems are difficult to manufacture in house and can be prohibitively expensive to obtain from commercial sources. The Leishmania tarentolae system offers a relatively cheap alternative which is capable of producing difficult to express proteins, but which is simpler to produce in large scale. However, this system suffers from batch‐to‐batch variability, which has been accepted as a consequence of the complexity of the extracts. Here we show an unexpected origin for the variability observed and demonstrate that small variations in a single parameter can dramatically affect expression, such that minor pipetting errors can have major effects on yields. L. tarentolae cell‐free lysate activity is shown to be more stable to changes in Mg2+ concentration at a lower ratio of feed solution to lysate in the reaction than typically used, and a higher Mg2+ optimum. These changes essentially eliminate batch‐to‐batch variability of L. tarentolae lysate activity and permit their full potential to be realized.