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Producing molecular biology reagents without purification.

ABSTRACT: We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

SUBMITTER: Bhadra S

PROVIDER: S-EPMC8168896 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Producing molecular biology reagents without purification.

Bhadra Sanchita S Nguyen Vylan V Torres Jose-Angel JA Kar Shaunak S Fadanka Stéphane S Gandini Chiara C Akligoh Harry H Paik Inyup I Maranhao Andre C AC Molloy Jenny J Ellington Andrew D AD

PloS one 20210601 6

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 3 ...[more]

PMID: 34061896

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Project description:The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8-33% overall yield with 90-98% purity despite the omission of HPLC purification.

Project description:Hydrogels have been broadly studied for applications in clinically motivated fields such as tissue regeneration, drug delivery, and wound healing, as well as in a wide variety of consumer and industry uses. While the control of mechanical properties and network structures are important in all of these applications, for regenerative medicine applications in particular, matching the chemical, topographical and mechanical properties for the target use/tissue is critical. There have been multiple alternatives developed for fabricating materials with microstructures with goals of controlling the spatial location, phenotypic evolution, and signaling of cells. The commonly employed polymers such as poly(ethylene glycol) (PEG), polypeptides, and polysaccharides (as well as others) can be processed by various methods in order to control material heterogeneity and microscale structures. We review here the more commonly used polymers, chemistries, and methods for generating microstructures in biomaterials, highlighting the range of possible morphologies that can be produced, and the limitations of each method. With a focus in liquid-liquid phase separation, methods and chemistries well suited for stabilizing the interface and arresting the phase separation are covered. As the microstructures can affect cell behavior, examples of such effects are reviewed as well. STATEMENT OF SIGNIFICANCE: Heterogeneous hydrogels with enhanced matrix complexity have been studied for a variety of biomimetic materials. A range of materials based on poly(ethylene glycol), polypeptides, proteins, and/or polysaccharides, have been employed in the studies of materials that by virtue of their microstructure, can control the behaviors of cells. Methods including microfluidics, photolithography, gelation in the presence of porogens, and liquid-liquid phase separation, are presented as possible strategies for producing materials, and their relative advantages and disadvantages are discussed. We also describe in more detail the various processes involved in LLPS, and how they can be manipulated to alter the kinetics of phase separation and to yield different microstructured materials.

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Producing molecular biology reagents without purification.

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Producing molecular biology reagents without purification.

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