Prokaryotic expression of antibodies

Arbabi‐Ghahroudi, Mehdi; Tanha, Jamshid; MacKenzie, Ross

doi:10.1007/s10555-005-6193-1

Cited by 164 publications

(88 citation statements)

References 189 publications

(160 reference statements)

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“…Bacteria can be used to display on their surface antibodies of different format for diagnostic applications [8,9] and to obtain elevated VHH productions in both the periplasm and the cytoplasm [10][11][12][13]. In contrast, the yields of reconstituted IgG-like molecules and fusions with some valuable tags remain low [14,15] due to either structural complexity or different redox requirements of the two partner polypeptides.…”

Section: Introductionmentioning

confidence: 99%

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

et al. 2014

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Background: The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space.

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Section: Introductionmentioning

confidence: 99%

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

et al. 2014

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“…Nanobody GFP is a single domain antibody fragment (VHH) derived from a llama antibody that binds to GFP with nanomolar affinity and has been described under several different names including GFP--binding protein 1 (GBP1), Enhancer and GFP--nanobody [29,36,37,42]. Solubility tags similar to His6--SUMO are known to facilitate high--level expression of nanobodies in the cytoplasm of E. coli [43]. Since oxidation of the single cysteine pair in the structure of the nanobody cannot normally take place in the reducing cytoplasm, we coexpressed sulfhydryl oxidase Erv1 and a disulfide isomerase DsbC from plasmid pMJS10 [30] together with His6--SUMO--Nanobody GFP in BL21--SI cells.…”

Section: Expression and Purification Of Functional Nanobody Gfpmentioning

confidence: 99%

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

Holmberg

Gowda

Andréasson

2014

Protein Expression and Purification

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A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.Protein Expression and Purification, http://dx.doi.org/10.1016/j. pep.2014.03.002 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. Permanent link to this version AbstractProduction of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single--gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET--based bacterial expression vector pSUMO--YHRC. The vector supports cloning for untagged expression as well as fusions to His6--SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single--step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. Protein expression/Protein purification/cloning/recombination cloning SUMO/nanobody/ Introduction Straightforward cloning and production of biologically active proteins is central for studies in biochemistry and biophysics. During recent years, several improvements in recombinant protein expression and purification techniques have been developed. Yet many laboratories still depend on unreliable cloning techniques based on ligation of endonuclease restriction products. Especially, when cloning multiple DNA fragments into one expression vector traditional methodology requires time--consuming reiteration of the process and the cloning step frequently becomes limiting in the protein production process. Current cloning methodology relies on PCR amplification of the relevant gene using two primers designed to facilitate the subsequent cloning step. Traditionally, unique restriction sites in the flanking primer regions are digested and the PCR product is ligated into a vector containing compatible cohesive ends. Similarly, in TA--cloning a single adenosine residue added at each end of the PCR product by Taq DNA polymerase facilitates ligation into a restricted vector carrying complementary thymidine residues [1]. An alternative to these methods is ligation independent cloning (LIC). In LIC flanking sequence extensions are added to the PCR product and are by enzymatic modification converted to single stranded overhangs that are annealed to a vector carrying complementary ends [2]. Cloning of multipl...

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“…On the contrary, ScFv can be readily produced in bacterial cells, in particular Escherichia coli (E. coli), often as inclusion bodies (IBs) [30][31][32][33][34]. When expressed as IBs, solubilization and subsequent refolding of the insoluble proteins are required for production of functional and soluble proteins.…”

Section: Introductionmentioning

confidence: 99%

Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

2012

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Monoclonal antibodies to the soluble antigens or cell surface markers hold great promise as effective human therapeutics. One of the major disadvantages is its large size, which prevents efficient penetration into the target tissues. Smaller version of antibodies, which has only antigen binding sites, is extensively investigated. It becomes increasingly apparent, however, that these smaller fragments of antibodies are rather difficult to produce, as the normally efficient mammalian secretion system does not work well for these fragments. Thus, refolding of insoluble proteins produced in Escherichia coli is a method of choice, although such refolding is mainly based on trial-and-error experiment. Here we describe a novel refolding system using a new amino acid-based detergent, N-lauroyl-L-glutamate, and arginine. This detergent appears to readily dissociate from proteins below critical micelle concentration (CMC), while remaining effective in protein solubilization above CMC. Arginine suppresses protein aggregation when the detergent concentration was reduced below CMC. The interaction of the detergent and arginine with proteins, which play an important role in protein refolding, will be discussed in great length.

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Prokaryotic expression of antibodies

Cited by 164 publications

References 189 publications

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

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