Correlation between in Vitro Stability and in Vivo Performance of Anti-GCN4 Intrabodies as Cytoplasmic Inhibitors

Wörn, Arne; Maur, Adrian Auf der; Escher, Dominik; Honegger, Annemarie; Barberis, Alcide; Plückthun, Andreas

doi:10.1074/jbc.275.4.2795

Cited by 128 publications

(135 citation statements)

References 52 publications

(67 reference statements)

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“…The Gal4 activation domain (amino acid residues 768 -881) was amplified by the PCR using pGAD424 (Clontech) as template. Both primers (upstream primer, 5Ј-CCA TGG GCC CAA GCT TTG CAA AGA TGG ATA AAG-3Ј; downstream primer, 5Ј-TTT GGG CCC GAA GAA CCG CCA CCA CCA GAA CCG CCT CCA CCA GAG CCA CCA CCA CCA GGC CTG ATC TCT TTT TTT GGG TTT GGT G-3Ј) contain an ApaI site suitable for cloning the Gal4 activation domain including the SV40 T-antigen nuclear localization signal N-terminal to the different scFvs in the context of pESBA-Act (15). The activation domain and the single-chain antibodies are separated by a (GGGS) 3 linker encoded by the downstream primer.…”

Section: Methodsmentioning

confidence: 99%

“…The center of spectral mass was calculated from the fluorescence spectra as described (25). The curves were normalized and analyzed for relative stabilities as described (15).…”

Section: Methodsmentioning

confidence: 99%

“…This reporter strain was named YDE173 and was used for growth selection and to quantify reporter gene activation by measuring ␤-galactosidase activity. The yeast strain YAdM2xGCN4-150, which expresses the enzyme ␤-galactosidase under the control of two Gcn4-binding sites, is described elsewhere (15).…”

Section: Methodsmentioning

confidence: 99%

“…Because the reducing environment of the cytoplasm prevents the formation of the highly conserved intradomain disulfide bridges, very stable frameworks are needed that fold in the absence of the disulfide bond and do not undergo aggregation (14,15). The sequence requirements that make an scFv very stable such that it might also function in an intracellular environment are only now emerging.…”

mentioning

confidence: 99%

“…The sequence requirements that make an scFv very stable such that it might also function in an intracellular environment are only now emerging. 2 We have recently described a model system to measure the binding activity of a series of cytoplasmically expressed scFvs in yeast (15). These scFvs, which carry the same CDR loops on different frameworks, were all directed against the dimerization domain of the sequence-specific transcriptional activator Gcn4 (16).…”

mentioning

confidence: 99%

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Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Maur¹,

Zahnd²,

Fischer³

et al. 2002

Journal of Biological Chemistry

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Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that singleframework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.Antibodies are pivotal components of the vertebrate immune system that can bind to almost any molecule with a high degree of specificity and affinity. These characteristics have been exploited to turn the natural antibodies into powerful biotechnological tools in diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation, cloning, and expression of the antibody genes in a wide variety of hosts (1-3). Several forms of antibodies have been constructed to obtain derivatives that carry the binding site in a smaller assembly. One of the minimal forms still retaining the full binding site is the single-chain Fv fragment (scFv) 1 (4 -6). In the scFv form, the variable regions of the heavy and the light chains, which bear the hypervariable loops (or complementarity determining regions (CDR)), are connected by a flexible linker, allowing the expression of the protein from a single cDNA sequence.Natural antibodies, which are secreted by plasma cells, have evolved to function in an extracellular environment. In contrast to full-length, double-chain antibodies, the scFv antibody form can in principle be readily expressed also in the cytoplasm of eukaryotic cells and directed to any compartment to target intracellular proteins and thus evoke specific biological effects. Hence, intracellular scFvs, also known as "intrabodies," might have a great potential in functional genomics by blocking or modulating the activity of a growing number of newly identified proteins, thereby contributing to the understanding of their functions. In the long run, intrabodies might even be used in therapeutic applications, possibly in gene therapy settings.However, there are only few examples of successful applications (7-13), and the cytoplasmic expression of scFvs is generally confronted with the difficultie...

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Section: Methodsmentioning

confidence: 99%

“…The center of spectral mass was calculated from the fluorescence spectra as described (25). The curves were normalized and analyzed for relative stabilities as described (15).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Maur¹,

Zahnd²,

Fischer³

et al. 2002

Journal of Biological Chemistry

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Yeast Surface Display

Lahti

Cochran

2009

Therapeutic Monoclonal Antibodies

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Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Edwardraja

Rameshkumar

Vijayaraj

et al. 2010

Biotech & Bioengineering

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Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition.

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Correlation between in Vitro Stability and in Vivo Performance of Anti-GCN4 Intrabodies as Cytoplasmic Inhibitors

Cited by 128 publications

References 52 publications

Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Yeast Surface Display

Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

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