Construction of an antibody phage display library with a XBBXBX CDR3 sequence

The phage display library was constructed to contain only scFv antibodies harboring the V_H3 dp-38 germline with an XBBXBX CDR3 amino acid sequence. Using the well-characterized HS4C3 scFv antibody from the Nissim library as template, the library was constructed in a series of 5 PCR procedures (PCR 1-PCR 5) (Fig. 1) [8, 13]. In all procedures, a general PCR mixture was used containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl₂, 0.5 μM primers (Table ​(Table1),1), 2.5 units Taq polymerase (Promega), and 0.75–1 mM dNTP in a total volume of 50 μL. The cycling temperatures were 5 min 95 °C followed by 25–40 cycles of 1 min at 95 °C, 1 min at 46–50 °C for annealing and 74 °C for 1.5–2 min for extension. Detailed PCR procedures are listed in Supplementary Table 1. In PCR 1, the V_H (without the CDR3 and framework 4) of the HS4C3 gene was amplified. In PCR 2, the V_L (including the c-Myc tag), the linker sequence and the framework 4 of the V_H of the HS4C3 gene were amplified. The PCR 3 procedure was performed to introduce a new CDR3 into the V_H gene using an 87 nucleotide degenerate reverse primer. This primer contained the CDR3 encoded as 5’-SNN NYK SNN NYK NYK SNN -3′, where S encodes for G or C; Y for C or T; K for G or T; and N for A, C, G, or T, to obtain the XBBXBX sequence (Table ​(Table1).1). Next, we combined the new V_H genes and the V_L gene in PCR4 using overlapping sequences (FR4 of the V_H). The product of PCR 4 was evaluated on 1% agarose gel and isolated using the QiaEX II agarose gel extraction kit (Qiagen GmbH, Hilden, Germany). The purified product was amplified in PCR 5. The final product was purified after 1% agarose gel electrophoresis using phenol/chloroform extraction. DNA was precipitated using ethanol, the pellet was washed with 70% ethanol and dissolved in 50 μL water. The final product was digested using NcoI and NotI restriction enzymes (Life Technologies BV, Breda, the Netherlands) and ligated overnight at 16 °C into a NcoI and NotI digested pHEN1 vector (a kind gift of prof. G. Winter, Cambridge) using the Sureclone Ligation Kit (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The ligation product was purified using phenol/chloroform extraction. The ligation product was transformed via electroporation into E. coli TG1 (K12, supE, hsdΔ5, thi, Δ(lac-proAB, F′(traD36, proAB⁺, lacI^q, lac-ZΔM15) electroporation competent cells, efficiency ≥1.0*10¹⁰ cfu/μg (Stratagene Cloning Systems, La Jolla, USA). To calculate the titer of transformants, dilutions of the original electroporated bacteria were plated onto 2xTY agar plates containing 0.1% (w/v) ampicillin and 1% (w/v) glucose and incubated overnight at 37 °C.

Schematic overview of PCR reactions used to construct the XBBXBX CDR3 antibody library. In PCR 1, the heavy variable chain (V_H) up to framework 3 (FR3) of the HS4C3 coding sequence was amplified. In PCR 2, framework 4 (FR4) of V_H and the light variable chain (V_L) of the HS4C3 coding sequence was amplified. The XBBXBX complementarity determining region 3 (CDR3) was engineered using the degenerate oligonucleotide CDR3 primer in PCR 3. The new V_H genes were combined with the V_L gene in PCR 4 and subsequently amplified in PCR 5. Note that the CDR3 primer contains, next to the CDR3 region, the FR4 region and part of the FR3 region. For primers, see Table ​Table1.1. scFv: single chain variable fragment antibody.

Oligonucleotide sequences used for construction of the XBBXBX CDR3 phage display library and sequencing of scFv antibody genes

^aS = [G,C], N = [A,G,C,T], Y = [C,T], K = [G,T]