Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems

Abstract

Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.

Fig 1. Schematic depicting enhanced cell-free protein synthesis from aggregating the intrinsic enzymes around NPs.

(CFPS) systems can suffer from limited reaction rates, likely due to diffusion between components as shown in the reaction to left. CdSe/ZnS core/shell quantum dots (QDs) bind the His-tag of some CFPS components and cross-link into NP-aggregates to bring them into proximity, potentially increasing the catalytic rates or product yield. The enzyme structures shown are known to be present in the CFPS utilized and are represented by structures drawn from the PDB. IDs: 1CRK (mitochondrial creatine kinase), 1FMT (E. coli methionyl-tRNA(f)Met formyltransferase), and, 3PCO (E. coli phenylalanine-tRNA synthetase) [–60].

Fig 2. Characterization of PURExpress®–QD conjugates.

(A) Chemical structure of the CL4 ligand used to make the QDs colloidally stable in aqueous shown in the open dithiol configuration. (B) Agarose gel electrophoretic mobility shift assay of 523 nm emitting CdSe/CdS/ZnS core/shell/shell QDs incubated without and with a series of decreasing concentrations of the PURExpress® protein solution. Less mobility is correlated with binding to enzyme and the magnitude of this is decreased as the protein solution is serially diluted. The dashed white line indicates the location of sample wells in the gel. (C) Left—High-resolution TEM micrograph of the 523 nm emitting CdSe/CdS/ZnS core/shell/shell QDs with an average diameter of 4.1 ± 0.5 nm. A single QD is circled in red for visualization. Right—High-resolution TEM micrograph of the 625 nm emitting CdSe/ZnS core/shell QDs utilized for nanoaggregation studies due to their larger size and higher electron density which makes for easier imaging. (D) TEM micrographs of the PURExpress® protein solution (i), 625 QDs in buffer (ii), and 625 QD mixed with 0.5× PURExpress® solution at two different magnifications (iii, iv). Only when the QDs are mixed with the PURExpress® solution is clustering seen. The grey shading around the QD clusters in (iii, iv) are believed to be the PURExpress® enzymes.

Fig 3. sfGFP production enhancement with QDs.

(A) Production of sfGFP fluorescence in arbitrary units over time for the optimal 75 nM QD concentration versus that of the “free” or QD negative reaction. Samples were excited at 485 nm and fluorescence monitored at 510 nm [69]. Plot for all the QD concentrations can be found in S1 Fig. (B) Yield of sfGFP, as estimated by average fluorescence from the end-range of the reactions, over the range of QD concentrations tested (red) and the free reaction (grey). ANOVA p-value < 0.05, F > F_crit, and Tukey-Kramer analysis, indicated free was statistically different than with QDs for all concentrations except for the 1 nM and 100 nM QDs reactions. The 75 nM QDs condition was significantly different than all other samples (alpha 0.05). For additional information on the statistical analysis, see S2 Fig and S1 Appendix.

Fig 4. Enhancement of functional PTE production by QDs.

(A) Reaction setup highlighting stopping of the CFPS reactions with kanamycin at different time points. Paraoxon hydrolysis tracked by measurement of the p-nitrophenol absorbance product. Schematic not to scale. (B) PURExpress® reaction with QDs produced functional PTE, the activity of which was monitored by absorbance. Kanamycin was added at various time points to quench translation. (C) Identical PURExpress® reaction without QDs treated in the same manner as panel (B) produced less functional PTE, resulting in less activity and p-nitrophenol product absorbance. PTE PDB ID: IPTA [76]. Other protein structures are the same as shown in Fig 1.

Fig 5. sfGFP production is enhanced with QDs in diluted PURExpress® reaction conditions.

(A) Production of sfGFP over time with a range of QD concentrations present versus a negative control as monitored by fluorescence. Samples were excited at 485 nm and fluorescence monitored at 510 nm [69]. Plot for all the QD concentrations can be found in S6 Fig. (B) Yield of functional sfGFP, as estimated by average fluorescence from the end-range of the reactions, over the range of QD concentrations tested (red) as compared to the QD-free reaction (grey). When tested, all samples were statistically different from the free reaction, see S7 Fig and S1 Appendix.