Replacing amino acids in translation: Expanding chemical diversity with non-natural variants

White, E. Railey; Reed, Timothy M.; Ma, Zhiyuan; Hartman, Matthew C. T.

doi:10.1016/j.ymeth.2012.03.015

Cited by 8 publications

(11 citation statements)

References 28 publications

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“…However, newer methods in generating inhibitors capable of targeting specific protein domains have made advances in recent years. Approaches that enable probing of large libraries of potential molecules, such as mRNA display [111,112] and fragment-based drug discovery coupled with NMR analysis [113–119], will aid in the quest to inhibit difficult structures like the PDZ domain. Initial molecules can then be altered in a series of rational approaches to targeting that could yield promising candidates for drug design.…”

Section: Expert Opinionmentioning

confidence: 99%

Targeting tumor invasion: the roles of MDA-9/Syntenin

Kegelman

Das

Emdad

et al. 2014

Expert Opinion on Therapeutic Targets

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Introduction Melanoma differentiation-associated gene – 9 (MDA-9)/Syntenin has become an increasingly popular focus for investigation in numerous cancertypes. Originally implicated in melanoma metastasis, it has diverse cellular roles and is consistently identified as a regulator of tumor invasion and angiogenesis. As a potential target for inhibiting some of the most lethal aspects of cancer progression, further insight into the function of MDA-9/Syntenin is mandatory. Areas covered Recent literature and seminal articles were reviewed to summarize the latest collective understanding of MDA-9/Syntenin’s role in normal and cancerous settings. Insights into its participation in developmental processes are included, as is the functional significance of the N- and C-terminals and PDZ domains of MDA-9/Syntenin. Current reports highlight the clinical significance of MDA-9/Syntenin expression level in a variety of cancers, often correlating directly with reduced patient survival. Also presented are assessments of roles of MDA-9/Syntenin in cancer progression as well as its functions as an intracellular adapter molecule. Expert opinion Multiple studies demonstrate the importance of MDA-9/ Syntenin in tumor invasion and progression. Through the use of novel drug design approaches, this protein may provide a worthwhile therapeutic target. As many conventional therapies do not address, or even enhance, tumor invasion, an anti-invasive approach would be a worthwhile addition in cancer therapy.

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Section: Expert Opinionmentioning

confidence: 99%

Targeting tumor invasion: the roles of MDA-9/Syntenin

Kegelman

Das

Emdad

et al. 2014

Expert Opinion on Therapeutic Targets

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“…PCR products can be used directly for the simultaneous expression of multiple proteins without laborious cloning and transformation steps (Gan and Jewett, ; Yabuki et al, ; Wu et al, ). CFPS platforms allow the addition of accessory factors that promote protein folding (Endo et al, ; Matsuda et al, ; Ozawa et al, ) or the incorporation of unnatural amino acids (Albayrak and Swartz, ; White et al, ). They also facilitate the expression of cytotoxic proteins that cannot be produced in living cells (Schwarz et al, ; Xu et al, ; Xun et al, ).…”

Section: Introductionmentioning

confidence: 99%

A versatile coupled cell‐free transcription–translation system based on tobacco BY‐2 cell lysates

Buntru

Vogel

Stoff

et al. 2015

Biotech & Bioengineering

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Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins.

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“…In coupled transcription/translation systems supplemented additionally with an appropriate RNA polymerase DNA templates can also be used. In contrast to traditional cell-based expression methods, CFPS offers shorter process times, limited protein hydrolysis and the ability to express toxic proteins or proteins containing specific chemical groups or unnatural amino acids at defined positions [2]. Furthermore, the open nature of the system allows the reaction to be controlled and monitored directly.…”

Section: Introductionmentioning

confidence: 99%

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

et al. 2014

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BackgroundCell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase.ResultsThe highly-productive BY-2 lysate (BYL) can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system.ConclusionsDue to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

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Replacing amino acids in translation: Expanding chemical diversity with non-natural variants

Cited by 8 publications

References 28 publications

Targeting tumor invasion: the roles of MDA-9/Syntenin

Targeting tumor invasion: the roles of MDA-9/Syntenin

A versatile coupled cell‐free transcription–translation system based on tobacco BY‐2 cell lysates

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

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