Abstract
Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method – Golden Gate assembly with a bi-directional promoter (GBid) – for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to “scFv-like” simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert – two variable chains and one bi-directional promoter – are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.
Highlights
The discovery and engineering of monoclonal antibodies using phage display usually utilizes one of two simplified display formats
An isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible bi-directional promoter cassette named BidP, composed of two opposite-facing tac promoters flanked by a pair of DsbAss cotranslational translocation signal peptides[18], was inserted between the hCH1 and hCLκ domains
We note that the bi-directional promoter cassette BidP is included in this vector only as a template for PCR amplification, as the BidP cassette contained in the vector is removed during Golden Gate incorporation of the VL-BidP-VH insert
Summary
The discovery and engineering of monoclonal antibodies (mAbs) using phage display usually utilizes one of two simplified display formats. PGBid, enables creation of Fab libraries via two simple steps – (i) a 3-fragment overlap extension PCR reaction to combine the central BidP with the two flanking variable domains, and (ii) a one-pot, one-fragment high-efficiency Golden Gate assembly reaction requiring only minimal amounts of enzyme to seamlessly incorporate the library insert into pGBid, without the need for separate restriction digestion or ligation reactions or agarose gel extraction of the input DNA This simple and scalable approach to the creation of Fab-displaying phage, referred to as Golden Gate with a bi-directional promoter (GBid), was validated by the incorporation of a functional HER2-binding Fab on the surface of M13 bacteriophage. The isolation of hCD20-specific mAbs from this library following four rounds of selection/counter-selection on transfected whole mammalian cells underscores the value of GBid for Fab/mAb discovery, even for difficult targets
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