Abstract
One of the main advantages of a cell-free synthesis system is that the synthetic machinery of cells can be modularized and re-assembled for desired purposes. In this study, we attempted to combine the translational activity of Escherichia coli extract with a heme synthesis pathway for the functional production of horseradish peroxidase (HRP). We first optimized the reaction conditions and the sequence of template DNA to enhance protein expression and folding. The reaction mixture was then supplemented with 5-aminolevulinic acid synthase to facilitate co-synthesis of the heme prosthetic group from glucose. Combining the different synthetic modules required for protein synthesis and cofactor generation led to successful production of functional HRP in a cell-free synthesis system.
Highlights
Recombinant DNA technology has enabled heterologous production of recombinant proteins, many proteins require more than ordered polymerization of amino acids to achieve a functional state
A lower temperature generally reduces the overall yield of protein synthesis, horseradish peroxidase (HRP) activity in the reaction mixture increased 3fold when the reaction temperature was shifted from 30°C to 20 °C
Under these conditions, ∼57% synthesized HRP was partitioned in the soluble fraction and exhibited 4-fold higher peroxidase activity than enzyme produced under standard reaction conditions (Figure 1)
Summary
Recombinant DNA technology has enabled heterologous production of recombinant proteins, many proteins require more than ordered polymerization of amino acids to achieve a functional state. While functional production of those recombinant enzymes requires co-production of prosthetic groups, related synthetic pathways may be nonnative in host cells. Due to its open nature, cell-free protein synthesis (CFPS) provides greater flexibility than cellbased gene expression methods. Customizing the chemical composition of the reaction mixture for CFPS has enabled the production of many proteins that are difficult to be expressed in the cytoplasm of cells (Kim and Swartz, 2004; Carlson et al, 2012; Jin and Hong, 2018). The early versions of CFPS systems suffered from low protein productivity and high reagent costs, recent advances in improving the energetics of ATP regeneration and methodologies of extract preparation have enabled the development of highly productive and economic CFPS systems
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