The low molecular masses of estradiol (272.38 Da) and progesterone (314.46 Da) make the measurement difficult: Since our measuring method SAW detects differences in mass and conventional antibodies are quite large (around 150 kDa), we needed the smallest possible antibodies - nanobodies. We decided to use this form of antibody due to the reason that nanobodies have only one tenth of mass of conventional antibodies and are also lighter than the alternative single-chain variable fragment (scFvs ). We want to detect mass changes with our chip and due to this nanobodies are more suited for our cause: The mass difference between bound and unbound nanobody is relatively larger (15 kDa to ~15,3 kDa) compared to bound and unbound antibody (150 kDa to ~150,3 kDa). Furthermore, nanobodies are not only lighter in weight, they are also significantly smaller than antibodies: 2-3 nm compared to approximately 10 nm of conventional antibodies , .
In a first step of in silico nanobody grafting, the amino acid sequences of both donor (10G6D6 and C12G11, respectively) and acceptor (3DWT) are numbered. There are different numbering schemes for immunoglobulin variable domains available . We decided to use the AHo numbering scheme, because placement of gaps is based on the spatial alignment of known three dimensional structures of immunoglobulin domains and not on sequence variability . The numbering was performed using ANARCI (SAbPred) (http://opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/anarci/). For further work, the numbered residues were transferred to an excel sheet. Both, the CDRs and the functional important framework residues of the donor (10G6D6 and C12G11) were combined with the acceptor (3DWT) and a new nanobody, the graft, was created.
Figure 1: Map of the grafted estradiol nanobody. The CDRs were taken from the mAb anti-estradiol antibody 10G6D6 and were subsequently grafted onto the scaffold – the nanobody 3DWT. For immobiliziation, a 2xHis tag was added. In accordance with our cloning strategy SfiI recognition sites were added at 5´ and 3´ ends as well as a 6 nt long appendix, because SfiI is an endonuclease which has low efficiency at terminal recognition sites.
Figure 2: Map of the grafted progesterone nanobody. The CDRs were taken from the mAb anti-progesterone antibody 15G12C12G11 and were subsequently grafted onto the scaffold – the nanobody 3DWT. For immobiliziation, a His tag was added. In accordance with our cloning strategy, SfiI recognition sites were added at 5´ and 3´ ends as well as a 6 nt long appendix, because SfiI is an endonuclease which has low efficiency at terminal recognition sites.
Table 1: Design of the estradiol nanobody. The first 44 amino acid residues (AHo numbering) of the donor antibody (10G6), the accepting antibody (3DWT) and the grafted estradiol nanobody are shown. The complementarity determining regions are highlighted in yellow.
Table 2: Design of the progesterone nanobody. The first 44 amino acid residues (AHo numbering) of the donor antibody (C12G11), the accepting antibody (3DWT) and the grafted progesterone nanobody are shown. The complementarity determining regions are highlighted in yellow.
Figure 3: Screenshot taken from ChimeraX analysing the estradiol graft. An alignment between the scaffold 3DWT and CDR donor (10G6D6) was performed to check for sterical impairments with the amino acid residues. Estradiol is depicted in yellow, the scaffold 3DWT in cyan and 10G6D6 is marked in grey. Furthermore, three grafting candidate CDRs of 10G6D6 are shown in red. Searching for impairments, we checked every amino acid residue for sterical clashes between the grafted amino acids and the remaining residues from the scaffold which are not neutralized by the grafting process.
Figure 4: Screenshot taken from ChimeraX analyzing the progesterone graft. An alignment between the scaffold 3DWT and CDR donor 15G12C12G11 was performed to check for sterical impairments with the amino acid residues. Depicted in white is progesterone. Highlighted in blue is the scaffold 3DWT and in red 15G12C12G11. Furthermore, shown in green are the three grafting candidate CDRs of 15G12C12G11. Searching for impairments, we checked every amino acid residue for sterical clashes between the grafted amino acids and the remaining residues from the scaffold which are not neutralized by the grafting process.
Furthermore, the sequences were optimized regarding their GC content, number and length of repeats.The created nucleotide sequences were purchased as gene synthesis from IDT and EurofinsGenomics. In general, the nanobody grafting process generates weak binders as the sequence of the scaffold is strongly modified, especially with regard to the CDRs, which can partly form new secondary structures. In addition, core frame work residues are also transferred from the donor to the scaffold, which changes its sequence, perhaps resulting in a changed structure of the scaffold in non-CDR areas. Therefore, the grafted nanobodies have to be improved by suitable affinity maturation measures .
Error-prone PCR (epPCR)
We tested the epPCR protocol with two polymerases, the OneTaq® DNA polymerase  and the Taq DNA polymerase  on an mRFP-encoding construct in the vector pJOE. To verify the influence of the generated mutations, we used the Q5® high-fidelity DNA polymerase as positive control. The proof-reading function of this enzymes leads to very low error rates in the amplification process . The mRFP construct was amplified according to the epPCR protocol and the amplification product was then cloned into pJOE via Gibson assembly. The resulting vector pJOE::mRFP was transformed into Escherichia coli DH5α cellsvia heat shock. Selection of successfully transformed cells was performed on LB-Agar plates with Amp50. IPTG was added to induce the promoter which controls the transcription of mRFP. After incubation overnight at 37 °C, the colonies were screened using colony PCR. Plates were incubated for one week at 4 °C to support protein folding
Figure 5: LB-Agar plate with E. coli DH5alpha pJOE::mRFP after incubation over night at 37°C and one week of incubation at 4°C. The mRFP was amplified using the Q5® high-fidelity DNA polymerase (NEB) using the standard protocol for Q5 DNA polymerase.
Figure 6: LB-Agar plate with Amp50 and IPTG of transformants of pJOE::mRFP after incubation over night at 37°C and one week of incubation at 4°C. The mRFP was amplified by epPCR using the Taq DNA polymerase for the random mutagenesis.
Figure 7: LB-Agar plate with Amp50 and IPTG of transformants of pJOE::mRFP after incubation over night at 37°C and one week of incubation at 4°C. The mRFP was amplified by epPCR using the OneTaq® DNA polymerase for the random mutagenesis.
The red colonies carry the mRFP construct with no or only minor mutations. White colonies are either colonies with an empty vector or they carry a vector with a mutated mRFP which encodes a non-functional mRFP. As shown in the figures above, usage of the epPCR protocol resulted in significantly more white colonies than red colonies compared to the control (see Fig. 4). It is also evident that some red colonies are still visible in the OneTaq sample (see Fig. 6). In the Taq Polymerase sample, hardly any red colonies are visible (see Fig. 5).
Figure 8: Gel image of the colony PCR. For each polymerase eight samples (5 µl each) were loaded, 100bp plus ladder used. The gel (1.2% agarose, 1x TAE) was run for 25 minutes at 100 V. The amplified mRFP has a size of 800 bp.
Figure 9: Gel image of the estradiol nanobody encoding fragment amplified by error-prone PCR. The numbers 1 to 20 depict the dilution round from which the sample was derived . A 100 bp plus ladder was used for size comparison. The gel (1.2% agarose, 1x TAE) was run for 25 minutes at 100 V. The amplified estradiol nanobody fragment is 400 bp large. The samples were run on two gels under the same conditions.
However, we had serious problems to amplify the sequence of the progesterone nanobody by epPCR. The epPCR was not successful even after several attempts.
Figure 10: Gel image of the progesterone nanobody encoding fragment amplified by error-prone PCR. The numbers 1 to 20 depict the dilution round the sample stems from. A 100bp plus ladder was used for size comparison. The gel (1.2% agarose, 1x TAE) was run for 25 minutes at 100 V. The amplified progesterone nanobody fragment is 400 bp large. The samples were run on two gels under the same conditions. A strong signal is only visible in the first two dilution steps. A weak signal can be seen until dilution 5, afterwards no bands can be seen anymore.
Therefore, we decided to purchase and use a commercial error-prone PCR kit from JenaBioScience . With this kit we were able to amplify the progesterone nanobody encoding fragment.
Figure 11: Gel image of the nanobody encoding fragments amplified by error-prone PCR (error-prone PCR kit). Five samples per nanobody were analyzed. A 100 bp plus ladder was used for size comparison. The gel (1.2% agarose, 1x TAE) was run for 25 minutes at 100 V. The amplified nanobody fragments are ~400 bp large.
Figure 12: Quality distribution of the reads from E2_S pre trimming. Analyzed with FastQC. Quality is measured with Phred score. Red area depicts a quality range from 0 to 20, yellow area from 21 to 28 and green area is a quality range from 29 to 38. The reads are represented by box plots: The average quality of the reads at each position is shown in the blue line. The median quality is represented by the red line. The yellow boxes indicate the quantiles and the extreme values are displayed by the black lines. It can be seen that the quality of the reads decreases with increasing length.
Figure 13: Quality distribution of the reads from E2_S post trimming. Analyzed with FastQC. Quality is measured with Phred score. The red area depicts a quality range from 0 to 20, yellow area from 21 to 28 and green area is a quality range from 29 to 38. The reads are represented by box plots: The average quality of the reads at each position is shown in the blue line. The median quality is represented by the red line. The yellow boxes indicate the quantiles and the extreme values are displayed by the black lines. It is evident that the quality improved significantly through processing.
After trimming of the raw data, the reads were mapped on the associated reference genome (estradiol nanobody or progesterone nanobody). The Mapping was done with the mapping aligner Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). The important mapping statistics are collected with the command flagstat of the tool samtools and are shown in the following table:
Table 3: Flagstats for the four sequenced samples E2_O, E2_S, E2_K and P_K. The data was retrieved using the “flagstat” command from samtools analysing the with Bowtie2 mapped data.
|Reads in total||733474||882572||886832||948928|
|Properly paired||726754 (99,85%)||872830 (99,87%)||829972 (99,43%)||912058 (99,52%)|
|Mapped reads||727879 (99,24%)||873962 (99,02%)||834762 (94,1%)||916458 (96,58%)|
Figure 14: Screenshot from the IGV file of the estradiol samples´ bam files, showing the coverage of the samples E2_O, E2_S and E2_K. Furthermore, deviations from the reference sequence (the initially grafted sequence of the estradiol nanobody) are shown and each base has a distinct colour: Adenine is shown in green, cytosine in blue, guanine in orange and thymine in red. The first paragraph depicts sample E2_O, second paragraph E2_S and the third paragraph E2_K.As shown in Figure 14, the two epPCR approaches show significantly more mutations than the control. Furthermore, an even distribution of the mutation sites is shown. The most noticeable feature of the IGV visualization are the two cytosines between base 200 and 250, which are present throughout all samples. These two mutations are also present in the control sample E2_O, where otherwise significantly fewer mutations occurred. This indicates that an error occurred during gene synthesis.
Figure 15: Screenshot from the IGV file of the progesterone sample bam file, showing the coverage of the samples P_K. Furthermore, deviations from the reference sequence (initial sequence of the progesterone graft) are shown and each base has a distinct colour: Adenine is shown in green, cytosine in blue, guanine in orange and thymine in red. The first paragraph depicts the sample P_K.
Figure 15 shows an even distribution of the mutation sites. The same two cytosines between base 200 and 250, which were present throughout all samples from the estradiol samples (E2_O, E2_S and E2_K), are present in the P_K sample as well. These facts strengthen the assumption that an error occurred during gene synthesis.
Table 4: Excel table created by the used python script, which shows the coverage of all reads per position. It also shows the percentage of each of the four bases per position in relation to the total number of reads. Shown are the first 10 of 410 lines of the excel sheet of the sample Prog_K.
Table 5: Mutation frequencies [bp-1] for the four sequenced samples.
|Sample||Mutation frequency [bp]-1>/sup>|
|E2_O||3,302 * 10-4|
|E2_S||1,992 * 10-3|
|E2_K||1,101 * 10-3|
|E2_P||1,283 * 10-3|
Figure 16: Graphical display of the calculated mutation frequencies [bp-1] of the “selfmade” and the kit epPCR amplification. Shown are the estradiol nanobody control sample, amplified by OneTaq DNA polymerase (E2_O, in grey); the estradiol sample generated by epPCR amplification using the protocol of McCullum et al. from 2010 (E2_S, in dark red) and the two samples of estradiol (E2_K, in light red) and progesterone (P_K, in green) amplified using the epPCR kit bought from JenaBioScience.
The mutation rate for the JenaBioScience epPCR kit is expected to be between 0,6 to 2% . In our experiments the kit yielded mutation rates per base pair of 1,101 * 10-3 for the estradiol nanobody and 1,283 * 10-3 for the progesterone nanobody, resulting in an average mutation rate per base pair of 1,192 * 10-3 per run of PCR. Although the “selfmade” epPCR protocol yielded a higher mutation rate per base pair we nevertheless chose the kit to generate our library from the nanobody fragments. This is because we could not achieve amplification of the progesterone nanobody by this protocol. That is why we chose the kit in the first place.
Figure 17: Overview of SfiI restriction sites. The gene of interest will be inserted between the pelB signal peptide (light pink coloured) and the linker (dark purple coloured), connecting it to the M13 geneIII protein. As insert is the grafted estradiol nanobody (grey coloured) and the immobilisation tag (green coloured) shown. A) Sequence of the 5´ SfiI restriction site with indicated cutting pattern. B) Sequence of the 3´ SfiI restriction site with indicated cutting pattern.
Figure 18: Map of the phagemid vector pZMB0062 with the sequence of the nanobody progesterone inserted.
Figure 19: Colony PCR of ER2738 clones after one day of incubation at 37 °C. Colonies 2, 3, 4, 7, 8, 9, 11 and 13 are positive for the progesterone nanobody insert. Colonies 1, 5, 10, 14 and 15 carry the religated vector without an insert. The gel (1,2% agarose with 1x TAE) was run at 100 V for 20 minutes. A 100 bp ladder was used for size reference.
Figure 20: Colony PCR of ER2738 clones after one day of incubation at 37 °C. The colony tested was positive for the estradiol nanobody insert. The gel (1,2% agarose with 1x TAE) was run at 100 V for 20 minutes. A 100 bp ladder was used for size reference.
For the detailed phage ELISA protocol click here.
A MaxiSorp 96-well plate was coated with streptavidin, subsequently biotin-coupled hormone was added to immobilize and present estradiol or progesterone. Incubation with our nanobodies presenting phages was followed by addition of anti-pVIII antibody with coupled HRP (horseradish peroxidase) and further addition of the substrate ABTS. Finally, the absorbance was measured in a Tecan i-control infinite 200 at 419 nm.
Table 6: Raw data (top) and corrected data (bottom) of the phage ELISA. The estradiol nanobody was tested against biotin conjugated estradiol in wells B4 to B6 and C4 to C6, well B7 served as control. The progesterone nanobody was tested in wells D4 to D6 and E4 to E6 against biotin conjugated progesterone, well C7 served as a control. The detection reaction was performed with the HRP conjugated anti pVIII antibody and ABTS substrate. Absorbance was measured at 419 nm in the plate reader Tecan i-control infinite 200.
The data (see Table 6) show no significant absorption at 419 nm, neither for the estradiol nanobody nor for the progesterone nanobody. Thus, it can be concluded that, as assumed and predicted, the initial grafts of the two antibodies do not bind their respective antigens or bind them poorly and that their specificity needs to improve by appropriate affinity maturation measures.
Figure 21: Visualization of the scFv Fragment for progesterone binding. The figure shows the overlap for Gibson Assembly at the 3’ and 5’ end of the fragment. Also the restrictionsites of SpeI and NdeI are shown.
Figure 22: Map of the pTXB1 vector with inserted scFv progesterone fragment.
The vector was used to overexpress our scFvs after successful cloning. Subsequently, the proteins can be isolated with the IMPACT™ Protein Purification System .
scFv Cloning Strategies
Cloning via Gibson Assembly®
1.1 After linearization of the backbone, a gel electrophoresis was performed in which the complete PCR product was inserted. The gel was then stained in ethidium bromide. The resulting fragments were visible on the transiluminator afterwards. With a scalpel the band was cut out of the gel at the ~ 6700 bp mark. The DNA was then recovered by using the Gel Clean up Kit from Machery-Nagel. The isolated DNA fragment and the scFv were then used for the Gibson Assemly®.
1.2 A second approach to preserve the opened backbone was the digestion of the PCR product by the restriction enzyme DpnI. Digestion with this restriction enzyme allows methylated template to be removed from the PCR, leaving only synthesized linearized plasmid DNA. After DpnI digestion, a PCR clean up was performed with the Machery-Nagel PCR and DNA clean up kit to remove debrist. The Gibson Assemly® followed.
Cloning via Seamless Cloning
Cloning via Restriction Enzymes
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