Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Abstract

Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. Besides, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNA_CUA and tRNA-synthetase pair, which could not be achieved in a living cell.

Keywords: Cell free system; Genetic code expansion; Simplified extract preparation; Thio-lysine.

Fig. 1

General scheme of the simplified preparation protocol and the modular approach for the cell-free reaction presented in this study. The entire protocol is divided into two phases: step, tools Phase one: bacterial growth is scaled up and induced to produce the desired components. Next, the cells are harvested, pelleted, and stored in the freezer. Phase two, the extract is prepared under the S30 protocol (vide infra) with small variations. Once several extracts are made, each containing different translational component pairs, the cell-free protein synthesis can be performed, where the modular reaction can produce one or more products while varying the components simply by mixing and matching extracts.

Fig. 2

Chemical structures of Uaas used in the present research. a) δ-thio-N-boc-lysine (TBK). b) N-propargyl-l-lysine (PrK). c) p-azido-l-phenylalanine (AzF).

Fig. 3

Comparison of performance using different extract preparation protocols. a) Cell-free production of WT GFP and genetically expanded Y35PrK GFP. Fluorescence intensity of produced WT and mutant GFP for four protocols: 1) S30 protocol 2) No intermediate inoculation, log phase maintained 3) No intermediate inoculation, stationary phase instead of log-phase added to final culture in small volume 4) Similar to 3, but added in large volume to final culture to obtain near-induction OD b) Cell-free production of WT GFP with extracts harvested at different OD₆₀₀. Original S30 protocol suggested bacterial harvest at OD== 2, compared to higher bacterial harvest at OD =3. The results are not significantly different (two-sided T-test, t = 1.57, df = 4, p-val = 0.19). c) Cell-free production of GFP for a comparison between different plasmids, using Pyl-OTS for UAG suppression. Fluorescence intensity of expressed protein from three different batches of WT GFP and Y35X plasmid preparations. Each plasmid type encoded had the same sequence but was purified independently.

Fig. 4

Cell-free production of double mutant GFP protein (Y35UAAD193UAG), using extracts mixtures of different ratios. Fluorescence intensity of double mutant production was compared to WT protein or double mutant without any Uaas (negative control). a) mixtures of OTS pairs and mixtures of OTS single components extract comparison for double mutant GFP production. A two-extracts mixture composed of AzF-OTS for UAA suppression and Pyl-OTS for UAG suppression, while a four-extracts mixture composed of the following extracts: 1) AzF synthetase 2) AzF tRNA for UAA suppression 3) PrK synthetase 4) PrK tRNA for UAG suppression. b) OTS pairs extract mixture, between AzF-OTS for UAA suppression and increasing quantities of Pyl-OTS for UAG suppression, at different ratios. c) Linear regression of extract dilution for double mutant GFP expression measured by GFP fluorescence. d) Lyophilized extracts and mixing ratios of AzF-OTS for UAA supression and Pyl-OTS for UAG supression: 1:1, black; 1:2, blue Extracts were rehydrated with varying volumes of water (t-test, ns represents p-val>0.05, * represents p-val = 0.02).

Fig. 5

TBK incorporation into proteins, using genetically expanded cell-free protein synthesis. a) Cell-free kinetics of GFP Y35X production in the presence of different TBK concentrations. b) Cell-free kinetics of RFP K15X production in the presence and absence of TBK. c) ESI mass spectra of purified RFP K15X with TBK, before and after boc-deprotection. d) Anti-ubiquitin Western blot of genetically expanded cell-free protein synthesis of the 36.4 kDa Ub-Intein-CBD construct. In the left panel: Cell-free reactions of incorporation of PrK and TBK in different concentrations. The right panel presents a comparison between TBK incorporated into two different branching sites, K48X and K63X, in the ubiquitin construct in the presence or absence of TBK, the bend in site 63 in the absence of TBK could be a result of stop codon readthrough.