Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection

Abstract

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

The Art of Exploring Diatom Biosilica Biomaterials: From Biofabrication Perspective

“…(A) Reproduced with permission. [118] Copyright 2016, American Chemical Society. (B) Reproduced with permission.…”

“…[101] Additionally, green fluorescent protein (EGFP) expressed in DB could differentiate between DB at different formation stages and assess the localization of recombination proteins. [118] One optimization involved repositioning the silica-targeting peptide Sil3T8 to the C-terminus of single structural domain antibodies (sdAb) to improve antigen access (Figure 10C), enhancing the DB's potential as an environmental biosensor. [120] Furthermore, using single-chain antibodies (scFv) in DB maintained high-affinity binding in the presence of pro-solvents, without requiring organic solvents or covalent cross-linking.…”

“…[118] One optimization involved repositioning the silica-targeting peptide Sil3T8 to the C-terminus of single structural domain antibodies (sdAb) to improve antigen access (Figure 10C), enhancing the DB's potential as an environmental biosensor. [120] Furthermore, using single-chain antibodies (scFv) in DB maintained high-affinity binding in the presence of pro-solvents, without requiring organic solvents or covalent cross-linking. By expressing antibody lgG's binding domain on T. pseudonana's surface, hydrophobic anti-cancer drugs could be encapsulated and targetedly delivered to tumor sites, effectively killing tumor cells while sparing healthy tissues.…”

“…The N-and C-terminal regions of the sdAbs are also noted in blue and red, respectively. (A) Reproduced with permission [118]. Copyright 2016, American Chemical Society.…”

Advanced Applications for Protein and Compounds from Microalgae

“…Microalgae represent an important biotechnology resource for the production of recombinant proteins for pharmacological applications. Successful results were reported by the genetic manipulation of microalgae for the production of antigens for vaccines, antibodies, immunotoxins, hormones, and antimicrobial agents [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 ]. Strategies and techniques have been developed for the exploitation of microalgae as expression systems for a number of advanced genetic toolkits [ 62 , 63 , 64 , 65 ].…”