Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity

Kumar, Sanjeev; Kalsi, Jatinderpal; Latchman, David S.; Pearl, Laurence H.; Isenberg, David

doi:10.1006/jmbi.2001.4534

Cited by 16 publications

(19 citation statements)

References 46 publications

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“…The binding activities of 3D8 scFv, VH, and VL dramatically decreased in parallel with ionic strength (Fig. 3), particularly for VH and VL domains, like other anti-DNA Abs (3,4,39). Therefore, the electrostatic interactions between basic residues of the proteins and phosphate backbones of DNA substrates might be a main stabilizing force, which is also supported by the protein bindings to both ss-and dsDNA substrates regardless of their specific sequences (Table 1).…”

Section: Figurementioning

confidence: 88%

Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity

Kim

Lee

et al. 2006

Journal of Biological Chemistry

103

152

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Section: Figurementioning

confidence: 88%

Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity

Kim

Lee

et al. 2006

Journal of Biological Chemistry

103

152

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“…Our studies suggest that the constituent domains of the antibody molecule may determine the level of protein expression. 40 Most of the Fabs with native or hybrid combinations of light and heavy chains that we cloned exhibited reasonable expression levels (5 -9 mg = l of culture). Interestingly, however, all the Fabs possessing the light chain of the anti-DNA antibody 33H11 including its native light -heavy chain combination, expressed poorly (10 -40 mg = l), suggesting that 33H11 light chain conferred poor protein expression.…”

Section: Protein Expressionmentioning

confidence: 87%

“…Full induction is achieved at 1 mM IPTG. 40 However, in our experience successful induction invariably results in a signi cant inhibition in the growth of the cell culture, suggesting that the expressed Fab protein is toxic to the bacterial cell. One quick way, therefore, to ensure that protein expression has occurred, is to compare the OD 600 of the cell culture before and after IPTG induction.…”

Section: Protein Expressionmentioning

confidence: 92%

“…34 However, success in obtaining large amounts of functional protein has been mixed, mainly due to the problem of some antibodies being inherently more dif cult to express than others, and of whether antibodies produced by this method fold appropriately. 35 Several attempts have been made to express scFv or Fab fragments of anti-DNA antibodies in bacterial expression systems 18,19,[36][37][38][39][40] and, although signi cant success has been achieved, the level of expression achieved was very variable and generally low. Several studies have focused on investigating the structure -function relationship of mouse antibodies via their over-expression in bacteria (reviewed in Stollar 19 ).…”

Section: Echerichia Colimentioning

confidence: 99%

“…For example, debris with its major constituent membrane proteins, was detected by dot blots, bound to Fab probably via membrane protein phosphate groups. 40 Following IPTG induction the rate of cell growth is reduced and debris is produced by virtue of Fab toxicity to the cell, resulting in cell lysis. One way to reduce such losses is to minimize cell lysis and the consequent cell debris produced.…”

Section: Expression Of Anti-dna Antibodiesðmentioning

confidence: 99%

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Molecular expression systems for anti-DNA antibodies—2

Kumar

Kalsi²,

Ravirajan

et al. 2002

Lupus

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Antibodies to double-stranded DNA are the best-known serological markers of systemic lupus erythematosus, and are closely associated with its renal pathogenesis. How these antibodies recognize DNA is not fully understood. An understanding of the relationship between the functional attributes of an antibody with the three-dimensional structure of its antigen-combining site would allow an insight into the rules that dictate auto-antibody-nucleic acid interaction and consequent pathogenicity of the autoantibody. Data from such studies could assist the development of novel drugs as an approach to specific therapies that can inhibit or disrupt protein-nucleic acid interactions. A full understanding of the binding specificities can be achieved only by experimental determination of detailed three-dimensional structure of these antibodies alone, and of their complexes with specific DNA antigens. A prerequisite of such a study is the ability to produce multimilligram quantities of the antibody protein. However, these antibodies are particularly difficult to express, probably due to their DNA-binding activity. This review attempts to focus on the recent developments on the over-expression of anti-DNA antibody fragments in heterologous cell expression systems and their purification to homogeneity that would in turn allow their structural studies via crystallization.

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Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

Fink

Vazulka

Egger

et al. 2019

Biotechnology Journal

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Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome‐integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon‐limited microbioreactor cultivations with yields of 0–7.4 mg Fab per gram of cell dry mass. Antigen‐binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid‐free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80–300% of plasmid‐based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.

show abstract

Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity

Cited by 16 publications

References 46 publications

Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity

Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity

Molecular expression systems for anti-DNA antibodies—2

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

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