A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans

Du, Ting; Buenbrazo, Nakita; Kell, Laura; Rahmani, Sadia; Sim, Lyann; Withers, Stephen G.; DeFrees, Shawn; Wakarchuk, Warren W.

doi:10.1016/j.chembiol.2018.10.017

Cited by 36 publications

(26 citation statements)

References 56 publications

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“…7(c)]. Although in this study the glycans were removed after cellular processing, these results still suggest that dimeric apo LAP can be effectively engineered for expression in simpler systems that do not support post-translational modifications, such as Escherichia coli, which could greatly increase the speed of therapeutic development (Harding & Feldman, 2019;Du et al, 2019). Additionally, since there were no conformational differences between the highmannose and complex LAP glycoforms, this suggests that increasing or modifying the branching type of glycosylation will not alter the apo LAP conformation either, which is one approach that has been employed to prolong the circulating half-life of protein therapies and improve their overall pharmacokinetic profiles (Perlman et al, 2003;Keck et al, 2008).…”

Section: Discussionmentioning

confidence: 86%

Structural insights into conformational switching in latency-associated peptide between transforming growth factor β-1 bound and unbound states

Stachowski

Snell

2020

Int Union Crystallogr J

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Transforming growth factor β-1 (TGFβ-1) is a secreted signalling protein that directs many cellular processes and is an attractive target for the treatment of several diseases. The primary endogenous activity regulatory mechanism for TGFβ-1 is sequestration by its pro-peptide, latency-associated peptide (LAP), which sterically prohibits receptor binding by caging TGFβ-1. As such, recombinant LAP is promising as a protein-based therapeutic for modulating TGFβ-1 activity; however, the mechanism of binding is incompletely understood. Comparison of the crystal structure of unbound LAP (solved here to 3.5 Å resolution) with that of the bound complex shows that LAP is in a more open and extended conformation when unbound to TGFβ-1. Analysis suggests a mechanism of binding TGFβ-1 through a large-scale conformational change that includes contraction of the inter-monomer interface and caging by the `straight-jacket' domain that may occur in partnership through a loop-to-helix transition in the core jelly-roll fold. This conformational change does not appear to include a repositioning of the integrin-binding motif as previously proposed. X-ray scattering-based modelling supports this mechanism and reveals possible orientations and ensembles in solution. Although native LAP is heavily glycosylated, solution scattering experiments show that the overall folding and flexibility of unbound LAP are not influenced by glycan modification. The combination of crystallography, solution scattering and biochemical experiments reported here provide insight into the mechanism of LAP sequestration of TGFβ-1 that is of fundamental importance for therapeutic development.

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Section: Discussionmentioning

confidence: 86%

Structural insights into conformational switching in latency-associated peptide between transforming growth factor β-1 bound and unbound states

Stachowski

Snell

2020

Int Union Crystallogr J

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“…In addition, innovative bioprocessing technologies and the application of scale-up methods for the production of recombinant proteins using E. coli have been established [ 136 , 229 ]. There are also novel endotoxin-free strains [ 233 , 234 ], strains that produce chaperones or that promote glycosylation [ 135 ]. Another achievement is the development of high-pressure refolding processes that promote antigen folding with characteristics similar to native virus proteins [ 235 , 236 ].…”

Section: Discussionmentioning

confidence: 99%

“…For example, only the glycosylated WNV E protein can optimally bind to C-type lectin DC-SIGN(R), a receptor that can either enhance the infection [ 133 ] or promote immune cell activation and antigen uptake [ 134 ]. Although bacterial cells do not usually promote such post-translational modifications, novel genetically modified E. coli strains that perform the glycosylation of recombinant proteins are good alternatives to deal with this problem [ 135 ]. Culturing conditions, solubility tags, and different expression vectors and strains can easily be evaluated in order to obtain a soluble antigen [ 136 ].…”

Section: West Nile Virusmentioning

confidence: 99%

Anti-Flavivirus Vaccines: Review of the Present Situation and Perspectives of Subunit Vaccines Produced in Escherichia coli

et al. 2020

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This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses.

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“…Recently, bacteria have also attracted great interest in their potential use for protein glycoengineering as a fast, simple, and low-cost expression system (Baker et al, 2013 ; Merritt et al, 2013 ; Yates et al, 2018 ). However, glycans in bacteria are also significantly different from human glycans ( Figure 5 ) (Du et al, 2019 ; Harding and Feldman, 2019 ). In order to circumvent the risk of immunogenic reactions from non-human glycans, several approaches to humanizing yeast and bacterial N-glycosylation pathways have been attempted over the last twenty years (Hamilton and Gerngross, 2007 ).…”

Section: Cell-based Protein Glycoengineeringmentioning

confidence: 99%

Protein Glycoengineering: An Approach for Improving Protein Properties

Guan

et al. 2020

Front. Chem.

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Natural proteins are an important source of therapeutic agents and industrial enzymes. While many of them have the potential to be used as highly effective medical treatments for a wide range of diseases or as catalysts for conversion of a range of molecules into important product types required by modern society, problems associated with poor biophysical and biological properties have limited their applications. Engineering proteins with reduced side-effects and/or improved biophysical and biological properties is therefore of great importance. As a common protein modification, glycosylation has the capacity to greatly influence these properties. Over the past three decades, research from many disciplines has established the importance of glycoengineering in overcoming the limitations of proteins. In this review, we will summarize the methods that have been used to glycoengineer proteins and briefly discuss some representative examples of these methods, with the goal of providing a general overview of this research area.

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A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans

Cited by 36 publications

References 56 publications

Structural insights into conformational switching in latency-associated peptide between transforming growth factor β-1 bound and unbound states

Structural insights into conformational switching in latency-associated peptide between transforming growth factor β-1 bound and unbound states

Anti-Flavivirus Vaccines: Review of the Present Situation and Perspectives of Subunit Vaccines Produced in Escherichia coli

Protein Glycoengineering: An Approach for Improving Protein Properties

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