Itoh K and Sokol SY (2014), Expression cloning of camelid nanobodies specif...

XB-ART-49482

PLoS One 2014 Jan 01;910:e107521. doi: 10.1371/journal.pone.0107521.

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Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

Itoh K , Sokol SY .

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Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

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Species referenced: Xenopus
Genes referenced: actc1 actg1 ctnnb1 krt7 nubp1 serpina6 vtga2
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	Figure 1. Expression cloning of nanobodies specific for Xenopus embryonic antigens. A scheme of the approach is shown. A nanobody cDNA library is made for bacterial expression using nested PCR from lymphocytes that were isolated from a llama immunized with Xenopus embryo lysates. Periplasm extracts prepared from pools of 50 bacterial colonies are used to stain embryo cryosections. Sib-selection of the positive pools leads to the identification of specific nanobodies. doi:10.1371/journal.pone.0107521.g001
	Figure 2. Immunostaining of gastrula embryos with pooled and individual nanobodies. Cryosections of Xenopus gastrulae were immunostained with periplasm (A, B) or individual nanobodies (C–E). A, Periplasm from pool 6 is a negative control. B, Periplasm from pool 8 shows punctate staining adjacent to the archenteron (Arch, arrow). C, NbP1 stains yolk platelets. D, NbP7 stains the cytoplasm and the cortex of both ectoderm and mesoderm cells. E. Gastrula section was costained with NbP7 (E′) and β-catenin (E′). Schematic insets indicate the approximate embryo region shown. Ecto, ectoderm, Meso, mesoderm, Endo, endoderm, BC, blastocoel. Scale bar is 50 µm. doi:10.1371/journal.pone.0107521.g002
	Figure 3. Monoclonal nanobody sequences and purification. A, Amino acid sequences of three isolated nanobodies are aligned. Two conserved cysteine residues and three complementarity-determining regions (CDRs) are indicated. B, Coomassie-stained gel demonstrating nanobody purification by Ni-chromatography. FT, flow through; E1–3, first to third elution; Ni, the Ni-resin before elution; peri, periplasm extract used for purification; BSA, bovine serum albumin. Asterisks indicate the positions of the purified nanobodies, NbP1 and NbP7. doi:10.1371/journal.pone.0107521.g003
	Figure 4. Immunodetection of specific antigens in embryo lysates. For western blot analysis, lysates of midgastrula (st. 10.5) and early neurula (st. 14) embryos were separated on SDS-PAGE gel, transferred to the Immobilon membrane and probed with different purified nanobodies, followed by anti-His tag antibodies. (A) NbP1 detects a major 47–49 kD protein and a minor 110 kD protein. (B) NbP7, but not NbP8, detects a 210 kD protein. β-catenin levels indicate protein loading. doi:10.1371/journal.pone.0107521.g004
	Figure 5. NbP1 immunoprecipitates a 49 kD protein from Xenopus embryos. A. NbP1 (P1) pull downs a specific protein from gastrula lysates (collected at st. 10.5–12). A normal embryo lysate (Lys) is shown on the left. NbP7 (P7) and no nanobody (-) groups represent negative controls. Immunoblot (probed with NbP1 and anti-His-tag) reveals heavy and light chains of the anti-His-tag antibody (asterisks), the nanobodies NbP1 and NbP7 (#) and the specific 47–49 kD protein band (arrowhead). B. Top candidate proteins detected by the LS-MS/MS analysis in a gel slice after immunoprecipitation by NbP1 from embryo lysates. Total scores based on the frequency of peptide representation, % coverage and molecular weights are indicated. doi:10.1371/journal.pone.0107521.g005
	Figure 6. Estrogen-regulated protein EP45/Seryp is a target of NbP1. Semiconfluent HEK293T cells were transfected with the indicated DNAs. The cells were lysed 24 hrs later, and the protein lysates were separated on SDS-PAGE for imunoblot analysis with NbP1, followed by an anti-His tag antibody. NbP1 detects EP45 (arrowhead) in lysates of cells expressing pCS2-Ep45, but not pCS2-GFP. Asterisk points to a nonspecific band, which reflects loading. Embryo (stage 11) lysate is on the right, with two visible bands, as a positive control. doi:10.1371/journal.pone.0107521.g006

References [+] :

Abler, Expression of scFv antibodies in Xenopus embryos to disrupt protein function: implications for large-scale evaluation of the embryonic proteome. 2003, Pubmed, Xenbase