scholarly journals A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam Chin-Fatt ◽  
Rima Menassa
Keyword(s):  
Fusion Proteins ◽  
De Novo ◽  
Molecular Farming ◽  
Passive Immunization ◽  
Limiting Factors ◽  
Expression Vectors ◽  
In Planta ◽  
Single Domain Antibody ◽  
Oxidative Folding ◽  
Thylakoid Lumen

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.

scholarly journals A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam Chin-Fatt ◽  
Reza Saberianfar ◽  
Rima Menassa
Keyword(s):  
Disulfide Bonds ◽  
Rational Design ◽  
De Novo ◽  
Passive Immunization ◽  
Fold Increase ◽  
Secretory Component ◽  
Secretory Iga ◽  
In Planta ◽  
Design Strategies ◽  
Single Domain Antibody

We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed ‘(5 + 1)Fc’, that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

scholarly journals Efficient In Planta Detection and Dissection of De Novo Mutation Events in theArabidopsis thalianaDisease Resistance GeneUNI

2016 ◽  
Vol 57 (6) ◽  
pp. 1123-1132
Author(s):  
Tomohiko Ogawa ◽  
Akiko Mori ◽  
Kadunari Igari ◽  
Miyo Terao Morita ◽  
Masao Tasaka ◽  
...  
Keyword(s):  
De Novo ◽  
De Novo Mutation ◽  
In Planta

Expression Vectors for Enzyme Restriction- and Ligation-Independent Cloning for Producing Recombinant His-Fusion Proteins

2008 ◽  
Vol 23 (3) ◽  
pp. 680-686 ◽  
Author(s):  
Blanca de las Rivas ◽  
José Antonio Curiel ◽  
José Miguel Mancheño ◽  
Rosario Muñoz
Keyword(s):  
Fusion Proteins ◽  
Expression Vectors ◽  
Ligation Independent Cloning

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

scholarly journals Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jan Weber ◽  
Zhaopeng Li ◽  
Ursula Rinas
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Limiting Factors ◽  
Expression Vectors ◽  
Metabolic Enzyme ◽  
E Coli ◽  
Metabolic Burden ◽  
Fructose 1,6 Bisphosphate

Abstract Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

scholarly journals Identification of client iron–sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana

2020 ◽  
Vol 71 (14) ◽  
pp. 4171-4187 ◽  
Author(s):  
Nathalie Berger ◽  
Florence Vignols ◽  
Jonathan Przybyla-Toscano ◽  
Mélanie Roland ◽  
Valérie Rofidal ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
De Novo ◽  
Bimolecular Fluorescence Complementation ◽  
In Planta ◽  
Chlorophyll Metabolism ◽  
Transfer Protein ◽  
Iron Sulfur ◽  
Bimolecular Fluorescence Complementation Assay ◽  
Client Proteins ◽  
S Proteins

Abstract Iron–sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

scholarly journals Preparation and Characterization of Anti-CeTNT-1 and Anti-CeTNT-4 Antibodies

1970 ◽  
Vol 44 (3) ◽  
pp. 371-376
Author(s):  
M Ziaul Amin ◽  
Hiroaki Kagawa ◽  
Mohammed A Satter
Keyword(s):  
Fusion Proteins ◽  
Tissue Specificity ◽  
Body Wall ◽  
Troponin T ◽  
Specific Interaction ◽  
The Body ◽  
Expression Vectors ◽  
Antibody Specificity ◽  
Sds Page

Among the four CeTNT isoforms, CeTNT-1, CeTNT-2 are body wall types, CeTNT-4 is pharynx type and CeTNT-3 is expressed in both the body wall and pharyngeal tissue. In our previous study, we used body wall and pharynx type anti-CeTNI, anti-CeTNC and anti-CeTM antibodies to observe the tissue specific interaction of the TNI isoforms with others TN subunits and tropomyosin isoforms. To extent the interaction study of CeTNT isoforms, in this study, we prepared and characterized the body wall type anti-CeTNT-1 and pharynx type anti- CeTNT-4 antibodies. For the preparation of the anti-CeTNT-1 and anti-CeTNT-4 antibodies, in this study we constructed the pCTNT-1 and pCTNT-4 expression vectors. The sub-cloned of the pCTNT-1 and pCTNT-4 expression vectors were verified by DNA sequencing. These expression vectors were used to generate fusion proteins of the body wall, TNT-1 and pharyngeal TNT-4 isoforms in Escherichia Ecoli. The expression of these fusion proteins were confirmed by SDS-PAGE analysis. The anti-CeTNT-1 and anti-CeTNT-4 antibodies were prepared in the rabbit by using the gel cut of the CeTNT-1 and CeTNT-4 fusion proteins. The antibody specificity of the CeTNT-1 and CeTNT-4 fusion proteins was also judged by Western-analysis using prepared anti-CeTNT-1 and anti-CeTNT-4 antibodies. The antibody specificity results indicated that anti-sera against each of both the body wall type TNT-1 and pharynx type TNT-4 isoforms had tissue specificity. Key words: Troponin T, Caenorhabditis elegans, Body wall, Pharynx DOI: 10.3329/bjsir.v44i3.4413 Bangladesh J. Sci. Ind. Res. 44(3), 371-376, 2009

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