Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

Amphipols (APols) are short amphipathic polymers that can substitute for detergents at the transmembrane surface of membrane proteins (MPs) and, thereby, keep them soluble in detergent-free aqueous solutions. APol-trapped MPs are, as a rule, more stable biochemically than their detergent-solubilized counterparts. APols have proven useful to produce MPs, most noticeably by assisting their folding from the denatured state obtained after solubilizing MP inclusion bodies in either SDS or urea. They facilitate the handling in aqueous solution of fragile MPs for the purpose of proteomics, structural and functional studies, and therapeutics. Because APols can be chemically labeled or functionalized, and they form very stable complexes with MPs, they can also be used to functionalize those indirectly, which opens onto many novel applications. Following a brief recall of the properties of APols and MP/APol complexes, an update is provided of recent progress in these various fields.

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“…NVoy also appears to provide an interesting medium for MP cell-free expression (Guild et al 2011; Klammt et al 2011). …”

Section: Applicationsmentioning

confidence: 99%

Amphipols for Each Season

2014

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“…Though the use of detergents and additives to refold the target protein can assist in making protein soluble, there is still no guarantee that these methods will be suitable for every protein of interest. When it comes to expression system, though a number of them, such as E. coli , Yeast, mammalian, insect cell, wheat germ cell-free expression system, and plant-based expression system (Goulding and Perry, 2003; Greene, 2004; Daly and Hearn, 2005; Guild et al, 2011), are available for protein production, the E. coli host system is widely regarded as the most suitable host for the expression of recombinant antibody fragments (Wang et al, 2008a, b). Compared to other host systems, the E. coli system is an economical, shows faster growth and is easier to manipulate genetically (Sushma et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Wang

Xiang

Feng

et al. 2013

Front. Cell. Infect. Microbiol.

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Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

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“…Over the past several decades, a variety of methods for producing a stable, soluble, and higher-affinity scFv antibody have been developed, including coexpression with molecular chaperons (3,31), expression in different host systems (32), genetic fusion to specific proteins (33), and GFP-based binders (29,30). Perhaps the most effectively used method is the use of a new type of antibody based on a GFP scaffold.…”

Section: Discussionmentioning

confidence: 99%

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Wang

Xiang

Zhang

et al. 2014

Appl Environ Microbiol

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Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

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Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

Cited by 21 publications

References 20 publications

Amphipols for Each Season

Amphipols for Each Season

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

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