Unexpected instabilities explain batch‐to‐batch variability in cell‐free protein expression systems. Issue 8 (20th April 2018)

• Record Type: Journal Article
• Authors: Hunter, Dominic J.B.; Bhumkar, Akshay; Giles, Nichole; Sierecki, Emma; Gambin, Yann
• Is Part Of: Biotechnology and bioengineering. Volume 115:Issue 8(2018)
• Page Start: 1904
• Page End: 1914
• Publication Date: 2018-04-20
• Subjects: cell‐free protein expression -- in vitro protein translation -- Leishmania -- Leishmania tarentolae -- LTE; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26604
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: 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 Mg 2+ concentration at a lower ratio of feed solution to lysate in the reaction than typically used, and a higher Mg 2+ optimum. These changes essentially eliminate batch‐to‐batch variability of L. tarentolae lysate activity and permit their full potential to be realized. Abstract : Leishmania cell‐free expression systems have always shown a high degree of instability and "batch‐to‐batch" variability. Here we show an unexpected origin for this variability, as we describe an instability in protein expression depending on a single parameter, the ratio of feed solution to cell‐free lysate. In the unstable zone, pipetting errors can translate into almost complete loss of expression. The picture shows the matrix of compositions we perform to map the metastable zone for each lysate and eliminate batch‐to‐batch variability.