High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

Lange, Stefan; Schmitt, Jutta; Schmid, Rolf D.

doi:10.1016/s0022-1759(01)00351-9

Cited by 45 publications

(30 citation statements)

References 33 publications

(33 reference statements)

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“…For example, quantities of 1 to 2 g of soluble, assembled FabЈ fragments/liter have been demonstrated in the periplasm of Escherichia coli (3). Secretion of ScFv fragments into the culture supernatant of Pichia pastoris at high titers, e.g., 250 mg/liter and 1.2 g/liter, has been reported by Eldin et al (6) and by Freyre et al (8), respectively, and up to 40 mg of secreted Fab/liter has been achieved with this yeast (16,25). In comparison to antibody fragments, there are very few reports of the production of full-length antibodies in microbial systems and, when reported, the titers have been very low (11,32).…”

mentioning

confidence: 76%

Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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Two different humanized immunoglobulin G1( ) antibodies and an Fab fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex 6 GlcNAc 2 to Hex 15 GlcNAc 2 . An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

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mentioning

confidence: 76%

Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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“…The advantage of this approach is avoidance of further processing of the antibody fragments by affinity purification for most applications in environmental analysis [58]. Antibody fragments are directly translocated into the culture medium by an inserted leader sequence.…”

Section: Choice Of Expression Hostmentioning

confidence: 99%

Recombinant antibodies for environmental analysis

Kramer

Hock

2003

Analytical and Bioanalytical Chemistry

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Initial steps of antibody engineering in the late eighties revolutionized the technology of antibody production, particularly in the area of immunotherapy and diagnostics. Hallmarks that seemed to be out of reach for a long time are now the state of the art, e.g. tailoring of antibodies to match particular needs or by-passing immunization by use of antibody libraries. Despite the apparent benefits of recombinant antibody technologies, this field has been opened up hesitantly for other applications. This review addresses the development of recombinant antibody synthesis in environmental analysis. Examples are given of the molecular evolution of pesticide antibodies and their application for the analysis of real samples.

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“…Once again, P. pastoris seems to be the host of choice, although some limitations became obvious. Functional Fab fragments could be efficiently secreted into the culture supernatant up to 40 mg/L in fed batch fermentations (Lange et al, 2001); however, only about 30% were correctly assembled to the heterodimer. Also Takahashi et al (2000) succeeded in Fab secretion driven by the P. pastoris phosphatase leader.…”

Section: Introductionmentioning

confidence: 99%

Engineering of Pichia pastoris for improved production of antibody fragments

Gasser

Maurer

Gach

et al. 2006

Biotech & Bioengineering

176

133

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The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor alpha (MFalpha leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion.

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High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

Cited by 45 publications

References 33 publications

Characterization of Humanized Antibodies Secreted byAspergillus niger

Characterization of Humanized Antibodies Secreted byAspergillus niger

Recombinant antibodies for environmental analysis

Engineering of Pichia pastoris for improved production of antibody fragments

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