Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins

Yao, Guangshuai; Huang, Chundong; Ji, Fangling; Ren, Jun; Luo, Xiuna; Zang, Berlin; Jia, Lingyun

doi:10.1039/d1cc07105j

Cited by 3 publications

(1 citation statement)

References 23 publications

(28 reference statements)

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“…These nanobodies can detect the discontinuous amino acids of a native protein structure thereby stabilizing it. Together, over-expressing an epitope EPEA tag (Glutamic acid-Proline-Glutamic acid-Alanine) bound to the recombinant protein and an anti-EPEA conjugated nanobody which is supposed to recognize each other thereby aiding in soluble protein production [ 59 ]. Our aim involved the use of a systematic design of experiments that tested the effect of salt, increasing buffer volume, adding a commercial solubilizing concoction (BugBuster ® ), using a mild detergent such as IGEPAL CA-630 and mechanical lysis using sonication.…”

Section: Discussionmentioning

confidence: 99%

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Shendge

D’Souza

2022

Microb Cell Fact

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Expression of affinity-tagged recombinant proteins for crystallography, protein–protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10–50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

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Section: Discussionmentioning

confidence: 99%

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Shendge

D’Souza

2022

Microb Cell Fact

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Nanobody-functionalized conduit with built-in static mixer for specific elimination of cytokines in hemoperfusion

Yao,

Ji,

Chen

et al. 2023

Acta Biomaterialia

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An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions

Ren

Xiong

Huang

et al. 2022

Journal of Chromatography A

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Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins

Abstract: Here we report a new protocol for enhancing the soluble expression of inclusion body (IB)-prone proteins in E. coli using nanobody (Nb) as a molecular-specific chaperone. The specific intracellular binding...

Cited by 3 publications

References 23 publications

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Nanobody-functionalized conduit with built-in static mixer for specific elimination of cytokines in hemoperfusion

An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions

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