Purification Systems Based on Bacterial Surface Proteins

Boström, Tove; Nilvebrant, Johan; Hober, Sophia

doi:10.5772/31078

Cited by 7 publications

(15 citation statements)

References 284 publications

(393 reference statements)

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“…Albumin-binding regions spanning one or several albumin-binding domains, for example BB and ABP [22] that are indicated in Figure 1, have been used for affinity purification or depletion of albumin [21]. Moreover, the use of an albumin-binding region as a fusion tag can facilitate affinity purification of a target protein, improve its solubility or be used for directed immobilization [22–24].…”

Section: Introductionmentioning

confidence: 99%

The Albumin-Binding Domain as a Scaffold for Protein Engineering

Nilvebrant

Hober

2013

Computational and Structural Biotechnology Journal

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The albumin-binding domain is a small, three-helical protein domain found in various surface proteins expressed by gram-positive bacteria. Albumin binding is important in bacterial pathogenesis and several homologous domains have been identified. Such albumin-binding regions have been used for protein purification or immobilization. Moreover, improvement of the pharmacokinetics, through the non-covalent association to albumin, by fusing such domains to therapeutic proteins has been shown to be successful. Domains derived from streptococcal protein G and protein PAB from Finegoldia magna, which share a common origin and therefore represent an interesting evolutionary system, have been thoroughly studied structurally and functionally. Their albumin-binding sites have been mapped and these domains form the basis for a wide range of protein engineering approaches. By substitution-mutagenesis they have been engineered to achieve a broader specificity, an increased stability or an improved binding affinity, respectively. Furthermore, novel binding sites have been incorporated either by replacing the original albumin-binding surface, or by complementing it with a novel interaction interface. Combinatorial protein libraries, where several residues have been randomized simultaneously, have generated a large number of new variants with desired binding characteristics. The albumin-binding domain has also been utilized to explore the relationship between three-dimensional structure and amino acid sequence. Proteins with latent structural information built into their sequence, where a single amino acid substitution shifts the equilibrium in favor of a different fold with a new function, have been designed. Altogether, these examples illustrate the versatility of the albumin-binding domain as a scaffold for protein engineering.

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Section: Introductionmentioning

confidence: 99%

The Albumin-Binding Domain as a Scaffold for Protein Engineering

Nilvebrant

Hober

2013

Computational and Structural Biotechnology Journal

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“…A). The advantages of choosing the ZZ‐peptide as a fusion protein include its small size, simple folding structure, high aqueous solubility, and the strong affinity for the Fc‐region of IgGs—a characteristic of Protein A itself (Boström et al, ; Braisted and Wells, ; Jendeberg et al, ). Consequently, the FAP‐ZZ reagents were expressed in Escherichia coli and purified via affinity chromatography utilizing the N‐terminal 6‐histidine tag (A).…”

Section: Resultsmentioning

confidence: 99%

Fluorogen‐activating‐proteins as universal affinity biosensors for immunodetection

Gallo

Vasilev

Jarvik

2013

Biotech & Bioengineering

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Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching.

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“…2 Protein A chromatography is a simple and highly selective method relying on the strong and specific interaction between Protein A and the crystallizable fragment (Fc) of the antibody. 3,4 Due to this, high yields and exceptional purity can be achieved. Alternatives to Protein A have been explored, such as cation exchange, [5][6][7][8] and nonchromatographic methods like precipitation, [9][10][11] but they have not been able to compete with the well-established Protein A platform when applied to industrial-scale bioprocessing.…”

Section: Introductionmentioning

confidence: 99%

“…Coupling degree (M)*Column volume 3. Number of binding domains per multimer 4. Amount eluted IgG/Molecules per column 5.…”

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confidence: 99%

Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

Scheffel

Kanje

Borin

et al. 2019

mAbs

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As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, ZCa, displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of ZCa to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, ZCaTetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with ZCaTetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.

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Purification Systems Based on Bacterial Surface Proteins

Cited by 7 publications

References 284 publications

The Albumin-Binding Domain as a Scaffold for Protein Engineering

The Albumin-Binding Domain as a Scaffold for Protein Engineering

Fluorogen‐activating‐proteins as universal affinity biosensors for immunodetection

Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

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