Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems

Verma, Rakesh; Boleti, Ekaterini; George, Andrew J.T.

doi:10.1016/s0022-1759(98)00077-5

Cited by 220 publications

(131 citation statements)

References 109 publications

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“…In this system, the protein posttranslational modifications are similar to those found in mammalian cells (Verma et al 1998). In addition, insect cells present several advantages when compared to mammalian cells, such as ease of culture, higher tolerance to osmolality (Olejnik et al 2003) and much higher expression levels when infected with recombinant baculovirus (Ikonomou et al 2003).…”

Section: *Corresponding Authorsupporting

confidence: 55%

Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture

Batista¹,

Pereira²,

Mendonça³

et al. 2006

Electron. J. Biotechnol.

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Section: *Corresponding Authorsupporting

confidence: 55%

Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture

Batista¹,

Pereira²,

Mendonça³

et al. 2006

Electron. J. Biotechnol.

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“…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%

“…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)(2)(3). Several forms of antibodies have been constructed to obtain derivatives that carry the binding site in a smaller assembly.…”

mentioning

confidence: 99%

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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“…The expression of several antibody fragments was evaluated in the BEVS and in stable transfected S2 cells. Reported maximum concentrations of the recombinant antibody fragments were 9 mg/L in the BEVS and 0.4 mg/L in S2 cells [23]. Production of recombinant human IL-7 in the BEVS and stable transfected Sf cells revealed in 10 times higher expression levels for the BEVS [24].…”

Section: Expression Of Two Different Proteins (Extracellular Vascularmentioning

confidence: 95%

Optimization of Insect Cell Based Protein Production Processes - Online Monitoring, Expression Systems, Scale Up

Druzinec

Salzig

Brix

et al. 2013

Yellow Biotechnology II

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Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems

Cited by 220 publications

References 109 publications

Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture

Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture

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

Optimization of Insect Cell Based Protein Production Processes - Online Monitoring, Expression Systems, Scale Up

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