Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) has the potential to produce enzymes, therapeutic agents, and other proteins, while circumventing difficulties associated with in vivo heterologous expression. However, the contents of the cell-free extracts used to carry out synthesis are generally not characterized, which hampers progress toward enhancing yield or functional activity of the target protein. We explored the utility of mass spectrometry (MS)-based proteomics for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions. Full proteome experiments identified over 1000 proteins per reaction. The complete set of proteins necessary for transcription and translation were found, demonstrating the ability to define potential metabolic capabilities of the extract. Further, MS-based techniques allowed characterization of the CFPS product and provided insight into the synthesis reaction and potential functional activity of the product. These capabilities were demonstrated using two different CFPS products, the commonly used standard green fluorescent protein (GFP, 27 kDa) and the polyketide synthase DEBS1 (394 kDa). For the large, multidomain DEBS1, substantial premature termination of protein translation was observed. Additionally, MS/MS analysis, as part of a conventional full proteomics workflow, identified post-translational modifications, including the chromophore in GFP, as well as the three phosphopantetheinylation sites in DEBS1. A hypothesis-driven approach focused on these three sites identified that all were correctly modified for DEBS1 expressed in vivo but with less complete coverage for protein expressed in CFPS reactions. These post-translational modifications are essential for functional activity, and the ability to identify them with mass spectrometry is valuable for judging the success of the CFPS reaction. Collectively, the use of MS-based proteomics will prove advantageous for advancing the application of CFPS and related techniques

rVλ6 amyloid fibrils cause metabolic dysfunction in cultured cardiomyocytes.

<p>Synthetic fibrils composed of rVλ6Wil (A), Jto (B), JtoR68S (C), and non-amyloid fibrils elastin (D) were shown to be fibrillar by using electron microscopy (left panels; bar = 100 nm; arrows indicate the region of fibrils shown inset). MTT reduction was measured in cultured AC10 human cardiomyocytes in the presence of fibrils (light) and monomeric proteins (dark) from 1 nM– 1 μM. *<i>p</i><0.05, for an unpaired t-test (right panel, n = 3). EM images were modified equivalently by increasing contrast and applying a shadowing effect, using Image J.</p

rVλ6Wil fibrils bind cultured cardiomyocytes.

<p>(A) Fluorescent synthetic fibrils composed of rVλ6Wil (Alexa Fluor-488, green; arrow) bound specifically to cultured AC10 cardiomyocytes (blue, nuclei; red, f-actin). (B) Confocal micrographs of cultured cardiomyocytes (blue, nuclei; purple, f-actin) after incubation with 1 μM fluorescent rVλ6Wil fibrils (green). Optical sections at the surface (Z-slice 20), center (Z-slice 45), and bottom (Z-slice 55) demonstrated the presence of fibrils on the cell surface (arrow) and rare intracellular material (arrowhead). All images were enhanced equivalently by increasing the brightness.</p

Metabolic dysfunction of cardiomyocytes is dependent on the presence of rVλ6Wil fibrils.

<p>(A) Supernatants from a suspension of rVλ6Wil fibrils generated by centrifugation of a fibril suspension at either 435,000 x g or 16,000 x g do not decrease MTT reduction by cardiomyocytes (supernatants were used at a 1x or 10x volume equivalent [vol. eq] to the unfractionated fibril suspension). Filtrate from a suspension of rVλ6Wil fibrils passed through a 100,000 (100K) MWCO filter did not contain material that affected MTT reduction (n = 3, per assay). (B) HPLC analysis of supernatant samples from the 435,000 x g (435K) and 16, 000 x g (16K) centrifugation as compared to the same volume of starting rVλ6Wil monomer (M) material. (C) Over the course of rVλ6Wil fibrillogenesis, only samples of the reaction mixture containing ThT-positive material significantly decreased MTT reduction when added for 24 h to AC10 cardiomyocytes. Gray circles, thioflavin T fluorescence (excitation = 450 nm; emission = 490 nm); black circles, MTT reduction (n = 3 sample per time point).</p