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.