{"id":1109,"date":"2019-05-17T14:58:49","date_gmt":"2019-05-17T06:58:49","guid":{"rendered":"http:\/\/www.bioactivescreeninglibrary.com\/?p=1109"},"modified":"2022-01-07T10:53:23","modified_gmt":"2022-01-07T02:53:23","slug":"high-protein-solubility-stability-camelid-vhh-domains-effective-translocation","status":"publish","type":"post","link":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/2019\/05\/17\/high-protein-solubility-stability-camelid-vhh-domains-effective-translocation\/","title":{"rendered":"High protein solubility and stability of camelid VHH domains are likely to help their effective translocation"},"content":{"rendered":"

In previous works, we have Epimedoside-A<\/a> studied the secretion mechanism of ATs and reported their capacity to translocate a model sdAb to the surface of E. coli fused to the \ufffd\ufffd-domain of ATs such as the IgA protease from Neisseria gonorrhoeae and EhaA from enterohemorrhagic E. coli O157:H7. A model sdAb fused to these \ufffd\ufffd-domains was correctly folded in the periplasm with the canonical disulfide bond of Ig domains formed by the action of DsbA. In a different study, we demonstrated that the Intimins from EHEC and enteropathogenic E. coli could be expressed in E. coli K-12 and displayed their native passenger domains on the cell surface with a disulfide bond formed by DsbA. However, neither the display of heterologous Ig domains fused to Intimin, nor the utility of the \ufffd\ufffd-domains of ATs and Intimin for display of sdAb libraries and de novo selection of sdAbs against an antigen of interest was investigated. In this work, we have demonstrated the capacity of the \ufffd\ufffddomains of EhaA and Intimin for the display of sdAb libraries on the surface of E. coli cells and for the selection of novel sdAbs against the extracellular domain of the translocated intimin receptor from EHEC. E. coli display libraries were screened by magnetic cell sorting and clones that specifically bound TirMEHEC were isolated, characterized by flow cytometry, as well as by enzyme linked immunosorbent assay and surface plasmon resonance with the purified sdAb. In addition, E. coli display was used to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions before protein purification. In this work, we have demonstrated that the \ufffd\ufffd-domains of EhaA and Intimin from EHEC O157:H7 are effective platforms for the display sdAb libraries on the surface of E. coli K-12 cells, and allow the selection of high affinity sdAbs from immune libraries using biotinylated antigen and MACS. Despite their opposite topologies, both systems express stable fusion proteins with the native \ufffd\ufffd-barrel correctly folded in the OM and display a functional sdAb with antigen-binding capacity on the surface of E. coli cells. Most sdAbs in the immune library were displayed on E. coli at good expression levels with both CEhaA and Neae, with an average between 6000-8000 molecules\/bacterium. From the immune libraries constructed in our study, we obtained a total of five independent camelid VHH sequences binding the antigen used in the immunization, being the sdAb of higher affinity and specificity the more frequent clone found in the selections with both \ufffd\ufffd-domains. These results demonstrate that both E. coli display systems can be used to retrieve high-affinity Ginsenoside-F4<\/a> binders from immune libraries of camelid VHH sequences. Fewer different binders were found compared to other reported studies using an immune library of camelid VHH displayed on the surface of phages and Staphyloccous carnosus cells. However, we think that the relatively small number of binders obtained in our study reflects a limitation of the anti-TirMEHEC library employed, and not of the E. coli-display systems described. Firstly, we have performed selections of the antiTirMEHEC VHH library on phage that failed to retrieve other specific binders than those selected by E. coli-display. Secondly, ongoing work in our laboratory with other immune VHH libraries also seem to indicate that essentially the same pool of binders can be isolated from phage and E. colidisplay systems. Thus, it appears that rather than the display system used, the actual diversity of binders retrieved from an immune VHH library is more dependent on factors such as immunogenicity of the antigen, number of animals used in the immunization, and library size.<\/p>\n","protected":false},"excerpt":{"rendered":"

In previous works, we have Epimedoside-A studied the secretion mechanism of ATs and reported their capacity to translocate a model sdAb to the surface of E. coli fused to the \ufffd\ufffd-domain of ATs such as the IgA protease from Neisseria gonorrhoeae and EhaA from enterohemorrhagic E. coli O157:H7. A model sdAb fused to these \ufffd\ufffd-domains … Continue reading “High protein solubility and stability of camelid VHH domains are likely to help their effective translocation”<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[],"_links":{"self":[{"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/1109"}],"collection":[{"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/comments?post=1109"}],"version-history":[{"count":1,"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/1109\/revisions"}],"predecessor-version":[{"id":1110,"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/1109\/revisions\/1110"}],"wp:attachment":[{"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/media?parent=1109"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/categories?post=1109"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.bioactivescreeninglibrary.com\/index.php\/wp-json\/wp\/v2\/tags?post=1109"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}