Ramping Recombinant Protein Expression in Bacteria

Zahurancik, Walter J.; Szkoda, Blake E; Lai, Lien B.; Gopalan, Venkat

doi:10.1021/acs.biochem.0c00411

Cited by 6 publications

(4 citation statements)

References 6 publications

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“…This discrepancy demonstrates that we still lack a complete picture of the subtle nuances that affect transcriptional and translational control. However, it is clear that the CDS considerably impacts gene expression and protein synthesis (Zahurancik et al ., 2020 ).…”

Section: Coding Sequencementioning

confidence: 99%

Importance of the 5′ regulatory region to bacterial synthetic biology applications

Tietze

Lale

2021

Microbial Biotechnology

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The field of synthetic biology is evolving at a fast pace. It is advancing beyond single-gene alterations in single hosts to the logical design of complex circuits and the development of integrated synthetic genomes. Recent breakthroughs in deep learning, which is increasingly used in de novo assembly of DNA components with predictable effects, are also aiding the discipline. Despite advances in computing, the field is still reliant on the availability of precharacterized DNA parts, whether natural or synthetic, to regulate gene expression in bacteria and make valuable compounds. In this review, we discuss the different bacterial synthetic biology methodologies employed in the creation of 5 0 regulatory regionspromoters, untranslated regions and 5 0 -end of coding sequences. We summarize methodologies and discuss their significance for each of the functional DNA components, and highlight the key advances made in bacterial engineering by concentrating on their flaws and strengths. We end the review by outlining the issues that the discipline may face in the near future.

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Section: Coding Sequencementioning

confidence: 99%

Importance of the 5′ regulatory region to bacterial synthetic biology applications

Tietze

Lale

2021

Microbial Biotechnology

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“…Translation efficiency is highly dependent on the mRNA secondary structures formed within the 5′UTR and between the 5′UTR and the start of the coding sequence (CDS), region referred to as the “gene ramp”, composed of the first 100 to 150 nucleotides , influencing translation initiation and protein yields. The gene ramp influences ribosomal entry onto mRNA and modulates early translation rates ,− (Figure a). The limitation in early translation rates at the 5′ gene termini was hypothesized to allow ribosomal spacing, therefore avoiding traffic jams, ribosome falloffs, and aborted translation. − The nucleotide composition of the ramp modulates the formation of mRNA secondary structures, in combination with the 5′UTR and within the ramp, that influence translation initiation kinetics .…”

Section: Introductionmentioning

confidence: 99%

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

et al. 2023

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Coordination of multigene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features of the 5′UTR in combination with the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures. These features strongly influence the translation yield and protein quality by regulating the ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5′UTRs in combination with different target genes, leads to a wide variety of gene ramp compositions with irregular translation rates, leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramp (SIGER) system for controlling protein expression. The SIGER system makes use of inteins to decouple the translation initiation features from the gene of a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid the impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways. As a proof of concept of the potential of the system, we also used a SIGER system to express two difficult-toproduce proteins, GumM and CBM73.

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“…Recombinant therapeutic protein production is a key aim of modern biotechnology due to huge market demand [ 1 , 2 , 3 ]. Recombinant protein expression systems have been established using various platforms including eukaryotic yeast, and mammalian and higher plant systems, but the most widely industrialised approaches involve prokaryotic bacteria [ 4 , 5 , 6 , 7 , 8 ]. These platforms have advantages and limitations in terms of expression efficiency, post-translational processing capacity, production cycle/costs, and application scope.…”

Section: Introductionmentioning

confidence: 99%

Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii

Wang

Zhuang

et al. 2022

Marine Drugs

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The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.

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Ramping Recombinant Protein Expression in Bacteria

Cited by 6 publications

References 6 publications

Importance of the 5′ regulatory region to bacterial synthetic biology applications

Importance of the 5′ regulatory region to bacterial synthetic biology applications

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii

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