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

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 concentration at a lower ratio of feed solution to lysate in the reaction than typically used, and a higher Mg optimum. These changes essentially eliminate batch-to-batch variability of L. tarentolae lysate activity and permit their full potential to be realized.

Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets with therapeutic potential

“…Alexandrov and sourced from Addgene (Addgene plasmid # 67137; http://n2t.net/addgene:67137; RRID:Addgene_67137). LTE extracts for in vitro expression were prepared in-house as described (55). Purified recombinant Mpro and PLP were generated by the UNSW protein production facility as described previously (14).…”

Single-molecule detection on a portable 3D-printed microscope

“…Leishmania tarentolae extracts (LTE) were produced as previously described in detail. 35,36 Plasmids containing Foldon in a cell-free expression vector (pCellFree_G10 37 ), which contains a C-terminal sfGFP 8xHis tag, as well as the sfGFP-containing vector itself, were transcribed and translated in LTE (at a final DNA concentration of 20 n m ). The reactions were incubated at 27 °C for 2 h and experiments were performed immediately after expression.…”

Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy

“…Cell-free expression systems provide a rapid and convenient approach for preparing proteins that are difficult to produce by other means. Here we used a Leishmania cell-free protein expression 31,32 system to produce GFP, GFP-CypA, and full-length GFP-CPSF6 (Supporting Fig S1A) and examined the interactions of these proteins with capsid in the Brightness assay ( Fig 2D, see Fig S1B for representative traces). The advantage of this system is that it allows rapid production of protein analytes (within ~2.5 h) in a microwell format in sufficient quantities for use in the assay without further purification.…”

“…The coding sequences for CypA, CPSF6 (isoform 2) and a fragment of NUP153 (residues 1407-1423) were cloned into the plasmid pCellFree_03 for expression with N-terminal GFP tag. 30 Each plasmid (60 nM) was added to Leishmania tarentolae extract 31,32 supplemented with RnaseOUT (1:1000, Invitrogen) and the mixture was incubated at 28 °C for 2.5 h.…”