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1. Cell-free systems for accelerating glycoprotein expression and biomanufacturing

   https://doi.org/10.1007/s10295-020-02321-4  

   Hershewe, Jasmine ; Kightlinger, Weston ; Jewett, Michael C ( November 2020 , Journal of Industrial Microbiology and Biotechnology)

   null (Ed.)

   Abstract Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines. 

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2. A Rapid and Facile Purification Method for Glycan‐Binding Proteins and Glycoproteins

   https://doi.org/10.1002/cpps.113  

   Welch, Christina J. ; Kadav, Priyanka D. ; Edwards, Jared L. ; Krycia, Jessica ; Talaga, Melanie L. ; Bandyopadhyay, Purnima ; Dam, Tarun K. ( September 2020 , Current Protocols in Protein Science)

   Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time‐saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour

   Alternate Protocol 1: Preparation of crude extracts from Jack bean meal

   Alternate Protocol 2: Preparation of crude extracts from the corms ofColocasia esculenta,Xanthosoma sagittifolium, and from the bulbs ofAllium sativum

   Alternate Protocol 3: Preparation ofEscherichia colicell lysates containing human galectin‐3

   Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin)

   Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension

   Basic Protocol 3: Detection of lectin activity of the crude extracts

   Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays

   Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays

   Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target‐TCA complex from other components of the crude extracts

   Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex

   Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents

   Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins)

    

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3. Engineering hydroxyproline‐ O ‐glycosylated biopolymers to reconstruct the plant cell wall for improved biomass processability

   https://doi.org/10.1002/bit.27266  

   Fang, Hong ; Wright, Tristen ; Jinn, Jia‐Rong ; Guo, Wenzheng ; Zhang, Ningning ; Wang, Xiaoting ; Wang, Ya‐Jane ; Xu, Jianfeng ( January 2020 , Biotechnology and Bioengineering)

   Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)‐O‐glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp‐O‐glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of “Ser–Hyp–Hyp–Hyp–Hyp” motif or (SP4)₁₈was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) fromAcidothermus cellulolyticus. The engineered (SP4)₁₈module was extensively Hyp‐O‐glycosylated with arabino‐oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5–11‐fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)₁₈module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)₁₈‐tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5‐fold without having adverse impact on the plant growth.

    

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4. Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins

   https://doi.org/10.1073/pnas.2107440118  

   Mingji Li, Xiaolu Zheng ( September 2021 , Proceedings of the National Academy of Sciences of the United States of America)

   As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein–protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties. 

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5. Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐ O ‐glycosylation process in plants

   https://doi.org/10.1111/pbi.13043  

   Zhang, Ningning ; Wright, Tristen ; Wang, Xiaoting ; Karki, Uddhab ; Savary, Brett J. ; Xu, Jianfeng ( December 2018 , Plant Biotechnology Journal)

   The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGPmodule secreted up to 56‐fold greater levels ofEGFP, compared with anEGFPcontrol lacking any HypGPmodule, supporting the function of HypGPmodules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈and (SP)₃₂modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fusedEGFPprotein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFPand (SP)₃₂‐EGFPintermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGPmodules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈module andAGP‐styled (SP)₃₂module is proposed.

    

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