Constructing and purifying megabodies starting from individual nanobody sequences

Megabodies are engineered nanobodies that help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interfaces. Here we describe how nanobodies can be rigidly grafted onto selected protein scaffolds (HopQ or YgjK) to increase their molecular weight while retaining the full antigen binding speci�city and a�nity. We also describe the protocols to purify these chimeric molecules from the periplasm of E. coli.


Introduction
Particle size, particle distribution in the vitreous ice layer greatly impacts on the quality and attainable resolution of cryo-EM reconstructions 1 .To help overcome these performance barriers, we designed a novel class of chimeric molecules, called megabodies (Mbs).Megabodies are built from a nanobody (Nb) that is grafted onto a scaffold protein via two short peptide linkers (Fig. 1a).Any nanobody can be converted into a megabody by linking the nanobody through its rst β-turn (connecting β-strands A and B) to an exposed β-hairpin of selected scaffold proteins.For this purpose, we have identi ed two large secreted bacterial proteins that are amenable to circular permutation and contain antiparallel βstrands with surface accessible β-turns: β-turn S3-S4 of the adhesin domain of H. pylori 2 (HopQ, 45 kDa, PDB ID: 5LP2, Fig. 2a) and β-turn A'S1-A'S2 of the E. coli K12 Glucosidase YgjK 3 (YgjK, 86 kDa, PDB ID: 3W7T, Fig. 2d).Here we describe how nanobodies can be grafted onto circular permutants of these molecular scaffolds to build megabodies of about 56 kDa (Fig. 1b) or 100 kDa (Fig. 1c), respectively using a single-step cloning procedure.We also provide protocols for the puri cation of milligram quantities of such megabodies from the periplasm of E. coli (Fig. 3).Produced megabodies retain the full antigen binding speci city and a nity of the parental nanobodies (Fig. 4).* The 3'-sequence of primer TU89 (TCCCTGAGACTCTCCTG) is the complement to the ORF encoding residues 18 to 23 (SLRLSC) of the consensus sequence of a nanobody (IMGT numbering).If the sequence of the parental nanobody is different in this conserved region, one can adapt the TU89 primer accordingly to maintain the sequence of the parental nanobody in the megabody.

REAGENTS SETUP
LB-ampicillin glucose plates.Dissolve 25 g of LB medium (high salt) and 15 g of agar in 900 ml of ddH 2 O. Autoclave the mixture and cool it to 50 °C.Supplement it with 100 ml of 20% (wt/vol) glucose and 1 ml of 100 mg/ml ampicillin (0.20-μm-lter-sterilized) and pour it into plates.The plates can be stored for one month at 4 °C.TB medium.Dissolve 12 g of Bacto tryptone, 2.3 g of KH 2 PO 4 , 12.5 g of K 2 HPO 4 , 24 g of yeast extract and 2.5 ml of glycerol in 1 liter of ddH 2 O. Autoclave the medium and store it for up to six months at 4 °C or for one month at RT. PE buffer.150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 8, and 20% w/v sucrose, 0.5 mg/ml lysozyme, 0.12 mg/ml AEBSF and 0.5 ug/mL leupeptin hemisulphate.Preparation: add lysozyme, AEBSF and leupeptin hemisulphate just before use.

Procedure
Reformatting nanobodies to megabodies Plasmids encoding megabodies for expression in E. coli can be constructed in a single step starting from any nanobody encoding gene according to Fig. 3. Gene fragments encoding β-strands B to G of the parental nanobodies (residues 18-128 according to the IMGT numbering, Fig. 3a) need to be ampli ed by PCR with primers TU89 and EP230.Ampli ed fragments are next cloned as SapI fragments in the desired expression vector: pMESD2 (GenBank MT328400) or pMESD22c7 (GenBank MT338520) to generate or megabodies (~56 kDa), and pMESP23E2 (GenBank MT338521) or pMESP23NO (GenBank MT338522) to generate or megabodies (~100 kDa), respectively (Fig. 3b-e).We recommend the (cloning vector pMESD22c7) and (cloning vector pMESP23NO) megabody formats for initial experiments.
Ampli cation of DNA fragments encoding nanobodies 1.To amplify a DNA fragment encoding the parental nanobody with two gene-speci c primers TU89 and EP230, combine the components and amplify DNA in a thermocycler as indicated in Table 1.
2. Analyze the PCR products by electrophoresis on a 1% (wt/vol) agarose gel.A DNA fragment of the parental nanobody ampli es as a ~350 bp fragment.Cut the 350-bp PCR product from a 1% (wt/vol) agarose gel and purify the fragment using the Wizard SV Gel and PCR Clean-Up System according to the manufacturer's instructions.Quantify the DNA from each puri cation by measuring the OD260.

Cloning nanobodies into megabody expression vectors of choice
Follow the restriction enzyme manufacturer's instructions to digest 4 μg of a megabody expression vector of choice with SapI and SalI in a 100-μl reaction mix.The supplementary digestion with SalI introduces additional non-complement digestion site and further reduces the sizes (< 20 bp) of two short fragments that are excised from the multi cloning site (MCS) to facilitate the puri cation of the SapI/SalI digested vector only with the Wizard SV Gel and PCR Clean-Up System, according to the manufacturer's instructions.
4. In parallel, digest 2 μg of the ampli ed DNA fragment of the parental nanobody (from Step 2) with SapI in a 50-μl reaction and purify the SapI digested DNA fragment with the Wizard SV Gel and PCR Clean-Up System. 5. Carry out a ligation reaction by ligating 100 ng of the SapI (/SalI) digested megabody vector (step 3) and a SapI digested ampli ed nanobody fragment (step 4) in 1:4 molar ratio, for 3 h at RT in a 10-μl reaction by using 0.5 units of the T4 DNA ligase.Heat inactivate at 65°C for 10 min.6. Transform 5 μl of each ligation into competent E. coli cells of choice (we recommend to directly transform expression strain WK6) by electroporation according to the manufacturer's instructions.Plate transformed cells on LB agar plates containing 100 μg/ml ampicillin and 2% (wt/vol) glucose and allow it to grow overnight at 37 °C.7. Pick 4 separate colonies and resuspend each colony in 50 μl of ddH 2 O and perform a colony PCR as indicated in Table 2. Analyze the PCR products by electrophoresis on a 1% (wt/vol) agarose gel.Clones that generate a fragment of ~600 bp have properly inserted the nanobody-encoding fragment within the MCS. 8. Inoculate a single PCR-positive colony in 5 ml of LB supplemented with 100 μg/ml ampicillin and 2% (wt/vol) glucose; grow overnight at 37 °C.Purify DNA plasmid using the QIAGEN Miniprep Kit, according to the manufacturer's instructions.Sequence puri ed DNA plasmids using primers listed in Table 2.
19. Pool the megabody-containg fractions obtained in step 16 and concentrate up to 500 µl using an Amicon® Filter Unit (10 kDa cutoff) rinsed with 1 ml of GF buffer.

Anticipated Results
In our hands, we were able to reclone any nanobody we tried so far in one of these megabody expression vectors to produce about 5-15 mg/L of the HopQ-or the YgjK-derived megabodies.These megabodies bind their cognate antigens with similar a nities as their parental nanobodies (Fig 4).Such megabodies can be stored for several days at 4°C and resist multiple freeze-thawing steps without proteolytic breakdown, precipitation or loss of antigen-binding capacity.

4.
Pardon, E. et al.A general protocol for the generation of Nanobodies for structural biology.Nat.Protoc.9, 674-693 (2014).Figure 4 Superdex™ 200 10/300 GL column, according to the manufacturer's instructions.21.Collect 0.5 ml fractions and analyze the content and the purity of each fraction using SDS-PAGE.Troubleshooting Time Taken • Ampli cation of DNA fragments encoding nanobodies: 1 day • Cloning into a megabody vector of choice: 4 days • Bacterial expression of 2 days • Release of megabodies from a periplasm and IMAC puri cation: 1 day • Gel ltration: 1 day

Figures
Figures

Figure 2 Structures
Figure 2