Efficient disulfide bond formation in virus-like particles

Bundy, Bradley C.; Swartz, James R.

doi:10.1016/j.jbiotec.2011.04.011

Cited by 92 publications

(95 citation statements)

References 49 publications

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“…50 Over the years, its utilization has expanded to include production of IgGs and antibody derivatives, 46 antibody-drug conjugates, 49 vaccines, 51 membrane proteins, 52 metalloproteins, 53 and viral proteins. 54 We report bispecific antibody expression, in particular KIH, using Xpress CF in this study. The high manipulability of the system 46,50,55 enables expeditious optimization of knob:hole plasmid ratio to achieve most efficient KIH assembly, as well as an "add-back" approach with prefabricated knob or hole for higher titer.…”

Section: Introductionmentioning

confidence: 88%

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Lee

Tran

et al. 2015

mAbs

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(2015) Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system , mAbs, 7:1, 231-242, DOI: 10.4161/19420862.2015.989013 To link to this article: https://doi.org/10. 4161/19420862.2015 Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering C H 3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

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Section: Introductionmentioning

confidence: 88%

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Lee

Tran

et al. 2015

mAbs

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“…More recently, eukaryotic chaperones such as Hsp70 or BiP (Welsh et al, 2011) have also being used. Bundy and Swartz (2011) have shown that it is possible to establish an oxidizing environment in the cell lysates that promotes disulfide bond formation through balancing the redox potential reaction.…”

Section: Future Perspectivementioning

confidence: 99%

Emerging technology of in situ cell free expression protein microarrays

et al. 2012

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Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUS-TER microarrays and the role of these methods in proteomics.

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“…In the last decade, cell-free systems have successfully been used to synthesize active disulfide-bonded proteins [51,52]. In the beginning of this development, two major hurdles had to be overcome: First, the presence of reducing agents, such as dithiothreitol (DTT) which were usually added in order to preserve the activity of the cell lysate during storage and the translation reaction itself and secondly, the lack of compartments with appropriate redox conditions as they are present in living cells (periplasmic space in prokaryotes, endoplasmic reticulum (ER) in eukaroytes) [53].…”

Section: Figurementioning

confidence: 99%

“…Depending on the source of the extract, different strategies have been applied to overcome these hurdles: For example, translation systems based on E. coli cell lysates were supplemented with defined mixtures of reduced and oxidized glutathione, in order to establish an appropriate and more oxidizing redox potential [52,[54][55][56][57]. Other translation systems focused on the preincubation of the lysate with alkylating reagents, such as iodoacetamide, in order to inactivate endogenous reductases which are present in the cell extract [51,52,57].…”

Section: Figurementioning

confidence: 99%

Cell-Free Synthesis Meets Antibody Production: A Review

Stech

Kubick

2015

Antibodies

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Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

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Efficient disulfide bond formation in virus-like particles

Cited by 92 publications

References 49 publications

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Emerging technology of in situ cell free expression protein microarrays

Cell-Free Synthesis Meets Antibody Production: A Review

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