Abstract
Easy preparation of chimeric nanobodies with various scaffolds is important for customizing abilities of nanobodies toward practical utilization. To accomplish high-throughput production of various nanobodies, utilization of microbes is an attractive option. In the present study, various chimeric nanobodies were prepared using the methylotrophic yeast Pichia pastoris. We designed chimeric nanobodies with complementarity-determining regions (CDRs) against green fluorescent protein (GFP) or cluster of differentiation 4 (CD4) based on the scaffold of GFP-nanobody. FLAG-tagged chimeric nanobodies were prepared by one-step cloning and produced using P. pastoris. Secreted chimeric nanobodies were purified from the culture media of P. pastoris transformants. Relative binding abilities of purified chimeric nanobodies to GFP and CD4 was tested using a BIACORE T-200. P. pastoris successfully produced a high yield of FLAG-tagged chimeric nanobodies. FLAG-tagged GFP- and CD4-nanobodies were shown to specifically bind to GFP and CD4, respectively. Chimeric nanobodies, in which the CDR2 or 3 of GFP-nanobody was replaced with CDRs of CD4-nanobody, acquired the ability to bind to CD4 without binding to GFP. These results demonstrate successful production of functional chimeric nanobodies using P. pastoris. These results also suggest that swapping of CDRs, especially CDRs 2 or 3, potentially enables a novel method of creating nanobodies.
Highlights
The heavy chain domains of Camelidae sp. antibodies (Hamers-Casterman et al 1993), or nanobodies, are increasingly attracting attention for their small size and ability to bind to targets
Relative binding abilities of chimeric nanobodies to cluster of differentiation 4 (CD4) CD4 and green fluorescent protein (GFP) were immobilized onto a CM5 chip at 9695.5 response unit (RU) and 1189.8 RU, respectively
To test the effect of complementarity-determining regions (CDRs) swapping on the ability of the nanobodies to bind to novel targets, relative binding abilities of chimeric nanobodies to CD4 was investigated (Fig. 4a and Additional file 1: Table S4)
Summary
The heavy chain domains of Camelidae sp. antibodies (Hamers-Casterman et al 1993), or nanobodies, are increasingly attracting attention for their small size and ability to bind to targets. Nanobodies are used in in vitro molecular studies, for example for isolating proteins and proteinassociated molecules (Bannas et al 2017). Being single-domain antibodies, nanobodies are produced by in vitro methods (McMahon et al 2018) or by various host cells including bacteriophages (Arbabi Ghahroudi et al 1997; Pardon et al 2014), Escherichia coli (Conrath et al 2001; Pardon et al 2014), yeast cells such as Aspergillus sp. According to comparison of different methods for producing nanobodies by Frenzel and De Meyer, optimized yield of nanobodies is generally similar among Gramnegative microbes, yeast cells, and mammalian cells (Frenzel et al 2013; De Meyer et al 2014). Microbial production of recombinant proteins is easier to test
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