Structural Characterization of Nanoyeast Single-Chain Fragment

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Structural Characterization of Nanoyeast SingleChain Fragment Variable Affinity Reagents Yadveer Singh Grewal, Muhammad J. A. Shiddiky, Lauren J. Spadafora, Gerard A. Cangelosi, and Matt Trau J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 12 May 2015 Downloaded from http://pubs.acs.org on May 13, 2015

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The Journal of Physical Chemistry

Structural Characterization of Nanoyeast Single-Chain Fragment Variable Affinity Reagents Yadveer S. Grewal, † Muhammad J.A Shiddiky, †* Lauren J. Spadafora, § Gerard A. Cangelosi§ and Matt Trau†ǂ*

Corresponding authors: Muhammad J.A Shiddiky*, Building 75, Cnr of College and Cooper Roads, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, ST LUCIA, QLD 4072 Australia. Tel: +61 7 3346 4178 Email: [email protected]

Matt Trau*. Building 75, Cnr of College and Cooper Roads, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, ST LUCIA, QLD 4072 Australia. Tel: +61 7 3346 4173 Email: [email protected]

†

Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and

Nanotechnology (AIBN), University of Queensland, Brisbane, Australia ǂ School of Chemistry and Molecular Biosciences, The University of Queensland, QLD 4072, Australia §

School of Public Health, University of Washington, Seattle, WA, USA.

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ABSTRACT Nanoyeast single-chain variable fragments (nanoyeast-scFv) are a new class of low-cost and stable protein capture agents developed as alternatives to full length monoclonal antibodies for use in immunosensors. Physical characteristics which imbue nanoyeast-scFv with these advantages have yet to been investigated. We investigate the structure, size and surface loading of nanoyeast-scFv to better understand its ability to specifically and sensitively capture proteins of interest while retaining activity in solution. Nanoyeast-scFv fragments were found to be globular in structure and heterogeneous in size, typically 100 nm sized structures in the nanoyeast layer (Figure 4 (B)) that is not at all evident in the underlying



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anti-HA layer (Figure 4 (A)). We propose these structures are nanoyeast-scFv fragments that are specifically captured by the underlying anti-HA layer, forming a larger aggregating structure. Addition of antigen reveals the nanoyeast-scFv layer with additional molecules that may be antigens bound to the surface (Figure 4 (C)). Similar observations were found when examining nanoyeast-scFv at higher (Figure S5) and lower (Figure S6) concentrations. Nanoyeast-scFv structures appeared consistent between different concentrations added (Figures 4, S5 and S6). Unsurprisingly, more nanoyeast-scFv was observed on substrates which had had higher concentrations of nanoyeast-scFv applied.

Figure 3. AFM micrographs of atomically flat glass substrates conjugated with (A) streptavidin/bio anti-HA, (B) streptavidin/bio anti-HA/nanoyeast-scFv, (C) streptavidin/bio antiHA/nanoyeast-scFv/ antigen ‘350’ at 1000 pg mL-1. Corresponding 3D topology of surfaces for


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(A') streptavidin/bio anti-HA, (B') streptavidin/bio anti-HA/nanoyeast-scFv, (C') streptavidin/bio anti-HA/nanoyeast-scFv/ antigen ‘350’ at 1000 pg mL-1. All micrographs presented as a 2x2 μm area. Colours indicate changes in surface height, with orange indicating neutral surface height of 0 nm, lighter colours indicating height increases compared to surface, and dark colours indicating a decrease of surface height compared to the neutral surface height. Scale bars = 0.5 μm.

Figure 4. FESEM micrographs of atomically flat glass substrates conjugated with (A) streptavidin/bio anti-HA, (B) streptavidin/bio anti-HA/nanoyeast-scFv, (C) streptavidin/bio antiHA/nanoyeast-scFv/ antigen '350' at 1000 pg mL-1. Scale bars = 100 nm.

Nanoyeast-scFv Structure in Solution. To investigate the structure of nanoyeast-scFv in solution, and the effects that filtering yeast debris has on the yeast particles in solution, nanoyeast-scFv were examined using transmission electron microscopy (TEM). Lyophilized yeast-scFv was resuspended in buffer and then examiined. Despite fragmenting lyophilized yeast with a mortar and pestle, many cells remained intact while other cells were ruptured and 15 ACS Paragon Plus Environment


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fragmented (Figure 5(A)). This finding suggests that the fragmentation process could be further refined to yield a higher number of fragments. Immungold labelling was performed on these fragmented yeast-scFv to attempt identification of yeast-scFv structures (Figure S7). Fragmented yeast-scFv were chosen for ease of sample processing due the larger structures present in fragmented yeast-scFv being better able to be collected for processing. Some level of specific binding appeared to evident to various yeast cell debris, however the results also showed some likely non-specific binding to the resin (Figure S7). Next yeast-scFv was filtered through a 100nm pore which excluded larger debris such as whole unfragmented cells. The remaining material (Figure 5(B)) varied greatly in shape and size, with most of the material lacking any definable structure. It is likely that the majority of nanoyeast fragments examined in this sample lacked scFv. Therefore, an affinity purification step is required to remove cellular material which lacks displayed scFv. Magnetic nanoparticles conjugated with anti-HA antibody were used to specifically capture nanoyeast scFv, allowing other material to be washed away. Molar excess of HA peptide was then added to gently elute the capture nanoyeast-scFv by competitive binding, resulting in a semi-purified sample of nanoyeast-scFv in solution (Figure 5 (C)). Dissolved nanoyeast-scFv purified by this process appeared to aggregate in micron sized structures. The individual components appeared to be globular in structure, with each structure ranging in size from 30 to 100 nm, consistent with our expectations of nanoyeast-scFv size. Aggregation of nanoyeast scFv could be attributed to the cellular components that form the yeast cell wall. Yeast cell wall is comprised of inner layer of structure supporting polysaccharides such as Chitin, 1,3 and 1,6--Glucans, with manoproteins located in the outer wall.16 These structures may aggregate in solution. However these same components are known help stabilise proteins, which could help attribute the stabilising properties of nanoyeast-scFv. It is therefore likely that


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nanoyeast fragments are comprised of the same polysaccharides which aid in providing stability to the associated yeast fragment. Indeed sugars have long been known to provide stability to proteins,17 with trehaolse - a disaccharide found in varying organisms, commonly used as stabilising agent for lyophilization of cells,18 and as a stabilising excipient for proteins.19 Wholecell yeast scFv are typically lyophilized (freeze-drying) for long term storage at room temperature. Prior work indicates these yeast-scFv reagents can be rehydrated and still retain activity.11 It is possible the yeast cell wall provides stability to these antibody fragments during the

lyophilized

process,

acting

as

inbuilt

stabilising

excipients.

Figure 5. TEM micrographs of (A) unfiltered nanoyeast-scFv (B) 100 nm filtered nanoyeastscFv and (C) 100 nm filtered and affinity purified nanoyeast-scFv. Scale bars are 1000, 100 and 200 nm respectively.

CONCLUSIONS Immunoassays require reagents that are robust enough to withstand demanding physical conditions such as long term storage at room temperature, yet also quick and cheap to generate. 17 ACS Paragon Plus Environment


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Nanoyeast-scFv possess these advantages. By retaining the scFv attachment to the yeast-cell wall, nanoyeast-scFv can be manufactured and used in an immunoassay within weeks, compared the months required for a mAb. Once manufactured as whole-cell yeast-scFv, they can be lyophilized and stored at room temperature for months, before being rehydrated and very simply and quickly fragmented, filtered and then used in an immunoassay within minutes. These advantages are due to allowing the scFv to be retained attached to fragments of the yeast cell wall - an environment for which it was selected to function over the course of FACS-based enrichment. In addition to speeding up the manufacturing process by removing the need to subclone and secrete the scFv from the yeast-display library, the yeast-cell wall provides stability to the associated scFv, likely due to the anchorage exerting constraints on the scFv structure. For the first time, we examined the physical characteristics of nanoyeast-scFv, and have demonstrated that sub 100 nm yeast-cell wall fragments are optimal for immunoassay performance. We also demonstrated that yeast fragments were heterogeneous in size and their structures were globular - possibly due to the polysaccharides which form the yeast cell wall. Unlike other antibody stabilising agents which prevent aggregation to provide stability,20 micrographs revealed aggregation of nanoyeast-scFv still occurred. This suggests that further refinement of the nanoyeast-scFv manufacturing process could still be performed, to prevent aggregation and further increase nanoyeast-scFv stability. That characterisations described here should help in understanding this new scFv format and how they best function as affinity reagent alternatives to full length mAbs in immunoassays.

Acknowledgment


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This work was supported by the NIH Grant, USA (U01AIO82186-01) and ARC DECRA. We acknowledge funding received by Trau's Laboratory from the National Breast Cancer Foundation of Australia via two National Collaborative Research Grants (CG-08-07 and CG-12-07). These grants have significantly contributed to the environment to stimulate the research described here. The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland, and Queensland node of the Australian National Fabrication Facility. We would like to thank Annie L. Becker for coordinating with the institutional review board for the ethics approval for the stool samples.

Supporting Information Available: AFM and cross-sectional height plot for nanoyeast-scFv conjugated onto a biosensor built onto an atomically flat glass substrate; AFM micrographs of nanoyeast-scFv at two different concentrations conjugated onto a biosensor comprised of a atomically flat glass substrate; Fluorescent microscopy images of biosensor on atomically flat glass substrate with secondary detection body and quantum dots showing the specificity of the assay; AFM micrographs of nanoyeast-scFv at two different concentrations conjugated onto a biosensor comprised of a atomically flat glass substrate; TEM immunogold labeling of fragmented yeast-scFv. This material is available free of charge via the Internet at http://pubs.acs.org.



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Structural Characterization of Nanoyeast Single-Chain Fragment

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