Nanobody Engineering and Detection through Directed Molecular Evolution and Microfluidic Screening...
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Nanobody Engineering and Detection through Directed Molecular Evolution and Microfluidic Screening
Our goal was to develop a protein engineering platform which is able to quickly and cost-effectively develop nanobodies with optimal binding affinity for any antigen of interest. These engineered nanobodies are desirable for protein therapeutics and diagnostic purposes.
Background Results
Alex Boone, Garrett Hovander, Xue Zou, Junyi Jiang, Kydric Luyen. PI: Prof. Xiaohua Huang
Research DesignInstrumentation Design
Microfluidic Screening Device
This is an integrated system that can be utilized for fluorescent and phase-contrast imaging of single molecules in our flow cell microfluidic screening device.
MBBS
MBPF
TL
Zeiss Axio Observer Microscope w/ autofocus & motorized XY stages (not shown)
Zeiss TIRF 3
Liquid light guide
Sutter DG5 (300 W Xenon)
488 nm
405 nm
532 nm
642 nm
EMCCDcameras
Dual-cam adaptor
BPF
Single-mode fiber Four lasers +Optical fiber
Dual-cam adaptor with2 Andor IXon3 EMCCDs
Andor IXon+ EMCCD
Sutter DG5
Ludl XY stagesZeiss autofocus
Zeiss TIRF3Slider
Zeiss Axio Observer
A manufactured microfluidic device can be used to screen nanobodies against novel antigens. Our proof of concept was demonstrated through the adherence of biotin-labeled antigens (α-Synuclein) that were covalently bound to a biotin, avidin coated glass substrate, allowing for our nanobody (NbSyn2) to successfully bind.
Nanobody Mutagenesis & Gene Synthesis
The nanobody moieties show the variable amino acid regions that were altered using directed mutagenesis.
The DNA stain shows evident 1kb gene synthesis of 10 oligonucleotides.
100 bases
1 kilobases
Primers
1kb GeneResearch Design
References
Conclusions
Acknowledgments
Biopharmaceuticals, also known as biologics, are terms used to refer to engineered macromolecular products such as proteins that may be used for medicinal purposes. Many antibody biologics are currently very expensive methods used to treat certain diseases. Not only are antibodies an expensive form of treatment, but their thermostability, accessibility to cryptic epitopes, and ease of manufacturability calls forth the need for a replacement protein biopharmaceutical. Thus, much research is being conducted in the field of nanobody synthesis. Nanobodies posses the aforesaid positive characteristics which allow for the generation of cheaper biopharmaceuticals which are overall more effective and cheaper for the patient. Through targeted directed mutagenesis, oligonucleotide synthesis may be performed in order to develop a high-quality DNA library for gene synthesis. Following gene synthesis, a ribosomal-display complex allows for the attachment of both mRNA and protein. This complex is achieved through the integration of a hairpin mRNA conformation that impedes the translation of mRNA. Therefore, the ribosome stays attached to both the mRNA and protein. By screening this complex at varying conditions (temperature) against a novel antigen, the mRNA within the ribosomal-display complex may be reverse transcribed to cDNA, amplified, and sequenced to determined genetic relationships.
CDR 2Asparagine-52; Valine-57; Lysine-58
CDR 3Phenylalanine-101; Serine-102; Tyrosine-105; Cysteine-106; Serine-109; Tryptophan-110; Serine-111; Asparagine-112
All the aforesaid mutation regions were determined through parallel camelid sequence alignment.
Gene Construct
Amino Acid Mutations
Following the synthesis of our gene, statistical analysis validated that our product had achieved a 2.4:1 fluorescence ratio of the 1kb to .2kb band synthesized by the T4 system. This yield was much greater than the Q5 system which merely yielded a 1:1 fluorescence ratio. Following the transcription and translation of our gene, we amplified our antigen product in E. Coli. Our antigen was purified and covalently bound to metallic beads. With real-time imaging, we were able to quantify the signal ratio between samples with the nanobody and samples without the nanobody. In conclusion, the samples that contained the engineered nanobody yielded approximately 1.5 times the signal than the samples that did not contain the nanobody. These results indicate that the engineered NbSyn2 mutated nanobody is viable for targeted binding.
Specific nanobodies have shown to be highly stable molecular proteins in extreme conditions (pH, temperature, etc.). As demonstrated, large libraries of mutated nanobodies may be artificially created for high-throughput screening. Furthermore, the microfluidics device is highly reliable since there are few factors to consider when performing these processes. These factors include the use of the appropriate antigen, DNA library, and thermal/chemical conditions. Our screening process is relatively simple with the use of a protocol for the biomolecular techniques in combination with the microfluidics device. Seeing that our results have shown enhanced favourable binding of our NBSyn2 nanobody to the Alpha-Synuclein antigen, there is promise of the capability of developing artificial nanobodies in order to target novel antigens.
1) S. Wiecek, Andrew. (2010). Nanobodies: Going single-domain. BioTechniques.2) Stefan Zielonka, et al, Shark Attack: High affinity binding proteins derived from shark cNARdomains by stepwise in vitro affinity maturation. J. Biotechnol. (2014)3) Douthwaite, Julie A., and Ronald H. Jackson. Ribosome Display and Related Technologies: Methods and Protocols. New York: Humana, 2012. Print.3) Douthwaite, Julie A., and Ronald H. Jackson. Ribosome Display and Related Technologies: Methods and Protocols. New York: Humana, 2012. Print.
We would like to recognize Dr. Xiaohua Huang and Dr. Pedro Cabrales for providing the resources and guidance to successfully complete this senior design research project.
Experimental Group: With Nanobody
Control Group: Without Nanobody