High level production of functional antibody fab fragments in an oxidizing bacterial cytoplasm11Edited by J. Karn

Venturi, Miro; Seifert, C.; Hunte, Carola

doi:10.1006/jmbi.2001.5221

Cited by 78 publications

(40 citation statements)

References 24 publications

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“…The further observation that DsbC was not required for cytoplasmic folding or subsequent Tat transport of scFv or Fab antibody fragments is consistent with these relatively simple pattern of disulfide bond formation in these two proteins. Indeed, disulfide isomerization does not appear to be a rate-limiting step in the folding and association of scFv or Fab fragments (29,48). In the present work, we report the construction of plasmid vectors for Tat translocation of recombinant proteins exhibiting more complex patterns of disulfide bond formation.…”

mentioning

confidence: 84%

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

Kim

Fogarty

et al. 2005

Appl Environ Microbiol

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When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.

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confidence: 84%

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

Kim

Fogarty

et al. 2005

Appl Environ Microbiol

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“…Although both the light chain and the heavy chain can be expressed separately as IBs, the refolding process was not eYcient and not economically competitive [32]. Expression of antibody fragments in an E. coli strain with an oxidizing cytoplasm also showed some promising results; however, this approach has not been adopted by the industry yet [116]. Periplasmic secretion is still the major route used by the industry for production of functional antibody fragments.…”

Section: Recent Advancements In Recombinant Therapeutics Productionmentioning

confidence: 99%

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Huang

Lin

Yang

2012

Journal of Industrial Microbiology and Biotechnology

369

248

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Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.

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“…[11][12][13] In alternative strategies, the optimization of individual antibodies was carried out in order to enhance the thermodynamic stability or the solubility and expression level of a particular molecule. [14][15][16][17][18][19] Such subsets of antibodies have been expressed in the bacterial cytoplasm in a stable and functional form, even though some residual aggregation or soluble aggregate formation may well occur.…”

Section: Introductionmentioning

confidence: 99%

Direct Selection of Antibodies from Complex Libraries with the Protein Fragment Complementation Assay

Koch

Gräfe

Schiess

et al. 2006

Journal of Molecular Biology

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High level production of functional antibody fab fragments in an oxidizing bacterial cytoplasm11Edited by J. Karn

Cited by 78 publications

References 24 publications

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Direct Selection of Antibodies from Complex Libraries with the Protein Fragment Complementation Assay

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