Amino acid balancing for the prediction and evaluation of protein concentrations in cell‐free protein synthesis systems

Abstract

Cell‐free protein synthesis (CFPS) systems are an attractive method to complement the usual cell‐based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well‐established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.

Cell-Free Protein Synthesis with Technical Additives – Expanding the Parameter Space of In Vitro Gene Expression

“…The average production of thscGAS-sfGFP under reference conditions with the inhouse CFPS system was 0.13 mg/mL, which is comparable to published data [12]. The fractional yield for thscGAS-sfGFP is about 10.5% and has therefore potential for optimization.…”

“…The fractional yield is the ratio between the theoretically achievable protein concentration based on the amount of provided amino acids in a CFPS system and the sequence of the target protein and the experimentally achieved result [12].…”

“…Fractional yields in this work have been calculated using the excel sheet provided by Rolf et al [12] and can be found in the supporting information.…”

“…Although CFPS has been used and improved since the 1960s, there are challenges in its application such as low production volume, batch-to-batch reproducibility, and reliable kinetic modeling of the system [10,11]. Furthermore, the transferability from CFPS screening results to the cells is limited but important as the in vivo production is often necessary for preparative scale applications [11,12]. So far, the description of CFPS systems focusses mainly on single components: the energy regeneration system, the cell extract itself, or individual buffer components [13,14].…”

“…In addition, we tested several organic solvents that are miscible and un-miscible with water. For the experiments, two model proteins, namely superfolder GFP (sfGFP) and the enzyme human cyclic GMP-AMP synthase-4 sfGFP, were used that differ in their size (sfGFP: 27 kDa, thscGAS-sfGFP: 84 kDa [2,16]) and fractional yield obtained for in vitro expression (sfGFP: 58%; thscGAS-sfGFP: 9% [12]).…”