title: A rapid and simple pipeline for synthesis of mRNA-ribosome-V(H)H complexes used in single-domain antibody ribosome display.
abstract: The single-domain antibody (VHH) is a promising building block for a number of antibody-based applications. Ribosome display can successfully be used in the production of VHH. However, the construction of the expression cassette, confirmation of the translation and proper folding of the nascent chain, and the purification of the ribosome complexes, remain cumbersome tasks. Additionally, selection of the most suitable expression system can be challenging. We have designed primers that will amplify virtually all Camelidae VHH. With the help of a double-overlap extension (OE) polymerase chain reaction (PCR) we have fused VHH with the F1 fragment (T7 promoter and species-independent translation sequence) and the F2 fragment (mCherry, Myc-tag, tether, SecM arrest sequence and 3' stem loop) to generate a full-length DNA cassette. OE-PCR generated fragments were incubated directly with cell-free lysates (Leishmania torentolae, rabbit reticulocyte or E. coli) for the synthesis of mRNA-VHH-mCherry-ribosome complexes in vitro. Alternatively, the cassette was ligated in pQE-30 vector and transformed into E. coli to produce ribosome complexes in vivo. The results showed that the same expression cassette could be used to synthesize ribosome complexes with different expression systems. mCherry reporter served to confirm the synthesis and proper folding of the nascent chain, Myc-tag was useful in the rapid purification of ribosome complexes, and combination of the SecM sequence and 3' stem loop made the cassette universal, both for cells-free and E. coli in vivo. This rapid and universal pipeline can effectively be used in antibody ribosome display and VHH production.
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Komenskeho 72, 04181 Kosice, Slovakia. bhidemangesh@gmail.com.
authors.forename: Elena
authors.initials: E
authors.lastname: Bencurova
authors.forename: Lucia
authors.initials: L
authors.lastname: Pulzova
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Flachbartova
authors.forename: Mangesh
authors.initials: M
authors.lastname: Bhide
mesh_terms: D000818:Animals; D002162:Camelus; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D000097573:Red Fluorescent Protein