Overview of the Purification of Recombinant Proteins

Wingfield, Paul T.

doi:10.1002/0471140864.ps0601s80

Cited by 151 publications

(95 citation statements)

References 183 publications

(196 reference statements)

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“…After curing each strain of the pORTMAGE plasmid, potential inhibitory effects on growth caused by the expression of tagged proteins were evaluated. Though the presence of the polyhistidine tags has previously been observed to cause growth defects due to the stability of tagged proteins, none of the cells produced for this work showed a significant drop in growth rate (Supplemental Figure 1) 29,30 .…”

Section: Preparation Of 6xhis-tagged Strains Using Magementioning

confidence: 80%

A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

Garcia

Dinglasan

Shrestha

et al. 2020

Preprint

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Highlights• A novel method of engineering crude cell lysates for enhancing specific metabolic processes is described. • Multiplex Automated Genomic Engineering (MAGE) can be used to engineer donor strains for improving cell-free metabolite production with minimal impact on cell-growth. • The described lysate engineering strategy can specifically direct metabolic flux and create metabolic states not possible in living cells. • Pooling of the central precursor pyruvate was significantly improved through use of this lysate proteome engineering strategy. AbstractCell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell's capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction.

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Section: Preparation Of 6xhis-tagged Strains Using Magementioning

confidence: 80%

A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

Garcia

Dinglasan

Shrestha

et al. 2020

Preprint

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“…Curiously, we observed significant batch-to-batch variation of ML6 activity between recombinant expressions (S6 Fig), which was not correlated to significant changes in lectin secondary structure (S7 Fig). In ongoing studies we are testing the sensitivity of ML6 to physical perturbations that may be induced during isolation and purification of the protein that potentially lead to the observed batch-to-batch variance, similar to other recombinantly expressed lectins [38].…”

Section: Anticancer Activitymentioning

confidence: 99%

Discovery of antitumor lectins from rainforest tree root transcriptomes

et al. 2020

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Glycans are multi-branched sugars that are displayed from lipids and proteins. Through their diverse polysaccharide structures they can potentiate a myriad of cellular signaling pathways involved in development, growth, immuno-communication and survival. Not surprisingly, disruption of glycan synthesis is fundamental to various human diseases; including cancer, where aberrant glycosylation drives malignancy. Here, we report the discovery of a novel mannose-binding lectin, ML6, which selectively recognizes and binds to these irregular tumor-specific glycans to elicit potent and rapid cancer cell death. This lectin was engineered from gene models identified in a tropical rainforest tree root transcriptome and is unusual in its six canonical mannose binding domains (QxDxNxVxY), each with a unique amino acid sequence. Remarkably, ML6 displays antitumor activity that is >10 5 times more potent than standard chemotherapeutics, while being almost completely inactive towards non-transformed, healthy cells. This activity, in combination with results from glycan binding studies, suggests ML6 differentiates healthy and malignant cells by exploiting divergent glycosylation pathways that yield naïve and incomplete cell surface glycans in tumors. Thus, ML6 and other high-valence lectins may serve as novel biochemical tools to elucidate the glycomic signature of different human tumors and aid in the rational design of carbohydratedirected therapies. Further, understanding how nature evolves proteins, like ML6, to combat the changing defenses of competing microorganisms may allow for fundamental advances in the way we approach combinatorial therapies to fight therapeutic resistance in cancer. OPEN ACCESS Citation: Lawanprasert A, Guinan CA, Langford EA, Hawkins CE, Sloand JN, Fescemyer HW, et al. (2020) Discovery of antitumor lectins from rainforest tree root transcriptomes. PLoS ONE 15 (2): e0229467. https://doi.org/10.

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“…Nonetheless, the following observations may guide the process. Protein samples can be synthesized in many different expression systems, but generally all will require purification of the target protein away from other components (Wingfield, 2015). Contaminating proteins not only confound analysis by outnumbering the protein of interest, contaminants can imperil the target protein's integrity via the presence of trace proteases.…”

Section: Commentary Background Informationmentioning

confidence: 99%

Negative‐Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging

et al. 2019

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Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative-stain image analysis to ascertain domain level molecular structure (10 to 20Å) more quickly and efficiently than possible by atomic resolution cryo-EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto-picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head-domain epitope. By comparing to the cryo-EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative-stain method. C 2019 The Authors.Keywords: electron microscopy r image analysis r image processing r negative stain

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Overview of the Purification of Recombinant Proteins

Cited by 151 publications

References 183 publications

A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

Discovery of antitumor lectins from rainforest tree root transcriptomes

Negative‐Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging

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