Creating the Nanobody Libraries for Phage Display

After purification, the antigens were used to immunize an alpaca to evoke an immune response and thus to generate novel affinity molecules. The immunization procedure and extraction of white blood cells was carried out by preclinics GmbH, Potsdam, Germany. Briefly, the frozen purified proteins (SNAP-25, syntaxin 1A, VAMP2) were shipped to the company and injected to the animal together with incomplete Freud’s adjuvant as described in section 2.2.14. After seven weeks, I received a

73

pellet of extracted white blood cells. The isolated blood cells were typically stored and shipped in RNAlater® to preserve the RNA.

In several trial extractions the conditions for the total RNA extraction procedure were optimized to minimize degradation and loss of sample quality. Isolation of peripheral B-cells from blood using Ficoll-gradient centrifugation rather than lysis of the erythrocytes turned out to yield best results (data not shown). For further preparations, the samples were shipped directly on the same day of extraction to the lab to minimize the time of transportation.

To isolate the total RNA from the cells, I used the RNeasy® Mini kit (Quiagen). To avoid diversity loss by saturation of the column, I used 8 columns per extraction providing a total capacity of 800 µg RNA. After extraction, the RNA was retro-transcribed using initially the SuperScript™ III and later the SuperScript™ IV system (Thermo Fisher Scientific). Although it was recommended to use random hexamer primers annealing to the RNA for reverse transcription, using directly site-specific primers for the IgG sequences significantly improved the cDNA yield about 10-fold. The first nanobody libraries were created by direct amplification of the nanobody sequences from IgG2 and IgG3 cDNA introducing peripheral restriction sites by PCR (Alp_F1+ and Alp_R1/2+ primers, see Table 17).

However, in subsequent experiments I realized that this method resulted in a rather low diversity of the library and thus in a limited number of specific nanobodies. Therefore I decided to set up a new approach omitting the inefficient cloning by restriction enzymes. All IgG heavy variable regions were first amplified in a nested PCR reaction (using the Call001 and Call002 primers) followed by specific amplification of nanobody sequences (using primers Gibson_F1 and Gibson_R1/2) as described by Pardon et al. [67]. At that second amplification step, 21 bp overhangs for Gibson assembly were introduced instead of the restriction sites. The phagemid was modified and amplified accordingly to allow insertion of the nanobody sequences by Gibson assembly resulting in an up to 20-fold higher cloning efficiency.

In various test reactions it turned out that the quality of TG-1 bacteria as well as the stoichiometric ratio of bacteria compared to plasmid concentration is crucial for an efficient electroporation. To maximize the efficiency of transformation, competent TG-1 bacteria were thus prepared freshly before each library generation. The medium used after electroporation was as well observed to have a great impact on the colony number. For the final libraries, I used Recovery Medium (Lucigen) to resuspend the electroporated cells, which resulted in a more than 10-fold higher number of colonies compared to SOC or LB medium. Table 21 provides an overview on different libraries for phage display created by electroporation.

Additionally, the ionic strength of the electroporation mixture was found to be very critical for the transformation. Ligated plasmid DNA therefore needed to be cleared from protein contaminants and eluted into pure water to minimize the salt concentration. A maximum of 1.5 µl of the ligation

74

reaction could subsequently be used to electroporate 50 µl of TG-1 bacteria without destroying the cells. As each TG-1 aliquot is further capable of internalizing only a limited number of plasmids, I empirically determined the best ratio of ligated plasmid versus bacteria not to exceed the competency of the bacteria. A maximum of 250 ng ligated plasmid DNA was used per reaction, which corresponds to ~150 fmol or 4.5 x 10¹⁰ molecules. Consequently, 40-50 individual electroporation reactions needed to be performed for each library as depicted in section 2.2.5.

A common way to determine the diversity is based on counting individual colonies after diluting the total library. With that, the diversity of my libraries ranges between 6.5 x 10⁶ clones for restriction-based and 4.5 x 10¹¹ for Gibson-based cloning as shown in Table 21.

Table 21: Overview on different libraries created for nanobody selection by phage display. The primer sets used to extract the nanobody sequences from total white blood cell preparations are indicated. To avoid overloading of the bacteria, successive electroporation reactions were performed using a maximum of 150 fmol DNA per reaction. The library diversity was estimated by counting individual colonies of plated bacteria dilution series after electroporation. After optimization the protocol, the amount of input RNA was increased to create the restriction-final library used for initial screen by phage display. As no novel nanobody families were revealed from the restriction library, I decided to create new libraries using Gibson cloning to maximize cloning efficiency and thus the sequence diversity in the library.

Library name RNA

75

Figure 13: Extraction of nanobody DNA sequences from alpaca IgG antibodies for cloning into phagemid vectors. A: Primer sets (black and red arrows) align to conserved elements of the IgG antibodies as described in Table 17 and Table 18. Degenerated primers were used to amplify the nanobody sequences in subsequent PCR reactions. Overhangs were added to the primers to create sites for enzymatic restriction or complementary sequences for Gibson cloning. B: Examples of PCR reaction to amplify the backbone vector (left) and the nanobody sequences (middle and right).

76