The cell-free protein synthesis (CFPS) system, as a technical core of synthetic biology, can simulate the transcription and translation process in an in vitro open environment without a complete living cell. It has been widely used in basic and applied research fields because of its advanced engineering features in flexibility and controllability. Compared to a typical crude extract-based CFPS system, due to defined and customizable components and lacking protein-degrading enzymes, the protein synthesis using recombinant elements (PURE) system draws great attention. This review first discusses the elemental composition of the PURE system. Then, the design and preparation of functional proteins for the PURE system, especially the critical ribosome, were examined. Furthermore, we trace the evolving development of the PURE system in versatile areas, including prototyping, synthesis of unnatural proteins, peptides and complex proteins, and biosensors. Finally, as a state-of-the-art engineering strategy, this review analyzes the opportunities and challenges faced by the PURE system in future scientific research and diverse applications.

Cell-Free PURE System: Evolution and Achievements

Abstract DHPS deficiency syndrome is an ultra-rare neurodevelopmental disorder (NDD) which results from biallelic mutations in the gene encoding the enzyme deoxyhypusine synthase ( DHPS ). DHPS is essential to synthesize hypusine, a rare amino acid formed by post-translational modification of a conserved lysine in eukaryotic initiation factor 5 A (eIF5A). DHPS deficiency syndrome causes epilepsy, cognitive and motor impairments, and mild facial dysmorphology. In mice, a brain-specific genetic deletion of Dhps at birth impairs eIF5A HYP -dependent mRNA translation. This alters expression of proteins required for neuronal development and function, and phenotypically models features of human DHPS deficiency. We studied the role of DHPS in early brain development using a zebrafish loss-of-function model generated by knockdown of dhps expression with an antisense morpholino oligomer (MO) targeting the exon 2/intron 2 (E2I2) splice site of the dhps pre-mRNA. dhps knockdown embryos exhibited dose-dependent developmental delay and dysmorphology, including microcephaly, axis truncation, and body curvature. In dhps knockdown larvae, electrophysiological analysis showed increased epileptiform activity, and confocal microscopy analysis revealed reduced arborisation of GABAergic neurons. Our findings confirm that hypusination of eIF5A by DHPS is needed for early brain development, and zebrafish with an antisense knockdown of dhps model features of DHPS deficiency syndrome. 

Deoxyhypusine synthase deficiency syndrome zebrafish model: aberrant morphology, epileptiform activity, and reduced arborization of inhibitory interneurons

Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes

In vitro translation systems are a useful biochemical tool to research translational regulation. Although the preparation of translation-competent cell extracts from mammals has often been a challenge, the commercially available rabbit reticulocyte lysate (RRL) is an exception. However, its valid use, investigating the mechanism of translation machinery such as ribosomes in RRL, presents an analytic hurdle. To overcome this issue, the hybrid translation system, which is based on the supplementation of purified human ribosomes into ribosome-depleted RRL, has been developed. Here, we describe the step-by-step protocol of this system to study translation driven by ribosomes lacking post-translational modifications of the ribosomal protein. Moreover, we combined this approach with a previously developed reporter mRNA to assess the processivity of translation elongation. This protocol could be used to study the potency of heterologous ribosomes.

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

/pdf/reconstitution-of-mammalian-mitochondrial-translation-system-5apcjtuab7.pdf

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA.

We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide.

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In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide.