To test this, we compared the influence of annealing efficiency under temperature cycling and non-cycling conditions. 400ng of VH and VL ssDNA template were mixed in a reaction tube and incubated. Agarose gel visualization of dsDNA formation under non-cyclic conditions showed a weak intensity band for the targeted 700 bp fragment with a stronger intensity band for the individual VH and VL ssDNA templates at 350 bp (Figure 5B). On the other hand, the band intensity after cycling steps showed a strong increase in the properly formed VH and VL hybrid templates (700 bp) with a coordinated reduction in the individual ssDNA templates (350 bp), indicating that cycling conditions greatly improve ssDNA annealing efficiency to form proper dsDNA (Figure 5C).
We concluded that for 400 ng of ssDNA template, temperature cycling is best for ssDNA template annealing followed by incubation at 37°C with 20 U of KF for 4 h to generate sufficient dsDNA template for cloning.
Diversity generation through chain shuffling
The ability to shuffle between heavy and light chains of an antibody is vital. Heavy chains play a crucial role in affinity, while light chains are more involved in antibody specificity (49). To demonstrate chain shuffling using our new ssDNA protocol, heavy and light chain primers were designed to give products with a complementary stretch of nucleotides and only a single 5′-phosphorylated end. As lambda exonuclease digests along the 5′-phosphorylated strands of both chains, this will lead to the sense strand of the heavy chain and antisense strand of the light chain remaining following enzyme treatment. Subsequently, the VH sense and VL antisense strands can anneal at the complementary region resulting in ssDNA templates of the scFv construct.
We performed our chain shuffling experiments as illustrated in Figure 2A. The VH and VL regions from anti-eGFP scFv clones 44 and G1 were amplified. Lengths of both fragments are approximately 400bp, and a negative control without the addition of the KF showed no band formation at 800 bp, while the sample with KF resulted in a band at the expected size (Figure 6A). The resulting 800 bp constructs then were ligated into the phagemid vector pIT2 and 10 clones were randomly selected and sequenced. All 10 clones sequenced showed successful exchange of the VL chains (Table 1).
By using our ssDNA and KF approach, we eliminated the primerless PCR step that is widely used in chain shuffling to get a full-length sequence. The primerless PCR step is cumbersome, as DNA smearing often occurs due to amplification of non-full-length sequences. The use of KF to generate full length sequences is easier, as it involves only one incubation step at 37°C (50).
Next we applied this protocol in the construction of a mini-library consisting of the heavy chain from anti-eGFP clone 44 and a natural pool of human kappa variable light chains (VK family 2, 4, and 6 genes) from human donors obtaining a clonal diversity of 2×108. The library size was estimated by a standard titration method (51). As the scFv heavy chain originates from a single clone, the only variability can be found in the light chain. Therefore, the method aims at producing new variants of a scFv with a given characteristic, such as binding to the same epitope on the target molecule with another affinity. A total of 12 clones were randomly picked and analyzed by DNA sequencing of both heavy and light chains. Ten clones (83%) showed successful introduction of new light chains from a human VK246 pool (Table 2).
le> Diversity generation with synthetic CDR3 oligonucleotides
To demonstrate another possible application of our directed evolution approach, we performed CDR3 mutagenesis where the 5′-phosphorylation was placed along the antisense primer binding to VH framework 3. The VH region was amplified by PCR and single strand templates were generated using lambda exonuclease according to the protocol described above. The synthetic CDR3 region is a 72-base antisense oligonucletide with a 9bp complementary region to VH framework 3 for annealing. Mutagenesis of CDR3 was accomplished through the incorporation of degenerate bases, as illustrated in Figure 2B. The resulting NNK degeneracy (where N represents a 25% use of adenine, thymine, guanine and cytosine nucleotides; and K represents a 50% mix of thymine and guanine) reduces the standard genetic code from 64 to 32 codons. It allows the coding of all 20 amino acids and eliminates 2 of 3 possible known stop codons (50). Next, the ssDNA of the VH and the synthetic CDR3 were mixed and extended using KF as described before. Agarose gel analysis demonstrated successful ligation as well as dsDNA formation of the CDR3 region with the VH backbone (Figure 6B).
The method was successfully used to generate a mini scFv library with a randomized CDR3 containing 6x107 clones. Out of the 27 clones selected for sequencing, 20 clones (75%) showed amino acid sequence variability in the CDR3 region (Table 3). All 4 degenerate positions along the CDR3 region were randomized without any repetition in the sequences of new hybrids. The measure of success for this newly developed protocol is the ability to generate a highly diverse collection of genes. Several assays such as Amplicot (52), DiVE (53) and DiStRO (54) have been developed to assay combinatorial nucleotide library diversity. The diversity of the generated CDR3 library was evaluated using DiStRO assay, which is a method designed to monitor nucleotide diversities based on DNA re-annealing kinetics (52). The re-melting curve from the assay is correlated to the diversity of a library. For a highly diverse library, more imperfect heteroduplexes are formed and the re-melting profile will be shifted. As shown in Figure 7, the re-melting curve of the CDR3 randomized library shows a left sided shift when compare with the control sample, anti-eGFP 44. The re-melting point of the library is around 80°C, which is within the range of the standard 12N library from DiStRO (54).
In summary, we demonstrate successful randomization at CDR3 using our new simplified directed evolution protocol, while framework sequences were unaffected and chain shuffling. This protocol allows regionally pre-determined randomization of genes. The success rate for the method is 75% to 83%, which falls well within range of other protein evolution methods, such as DNA shuffling (84%) (3), synthetic shuffling (72%) (16) and StEP (84%) (17).
The work was supported by the Malaysian Ministry for Higher Education through the Higher Institution Centre of Excellence (HICoE) Grant (Grant No. 311/CIPPM/44001005), Research University Cluster Grant (Grant No. 1001/PSKBP/8630015) and USM Short Term Grant Scheme (304/CIPPM/6310077). BN Lim would like to acknowledge financial support from the Malaysian Ministry of Higher Education MyBrain Program.
The authors declare no competing interests.
Correspondence Address correspondence to Theam Soon Lim, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Email: [email protected]">[email protected]References
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