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. 2018 Apr 25;14:83-88.
doi: 10.1016/j.bbrep.2018.04.005. eCollection 2018 Jul.

Surface Plasmon Resonance (SPR) Based Binding Studies of Refolded Single Chain Antibody Fragments

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Free PMC article

Surface Plasmon Resonance (SPR) Based Binding Studies of Refolded Single Chain Antibody Fragments

Pranveer Singh. Biochem Biophys Rep. .
Free PMC article

Abstract

Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as Escherichia coli has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and refolding protocol of single chain fragment variable (ScFv) and single chain antigen binding fragment (ScFab) using E.coli expression system. Efficiency of refolding protocol was validated by structural analysis by CD, native folding by fluorescence and functional analysis by its binding with full length HIV-1 gp120 via SPR. Results show the predominant β-sheet (CD) as secondary structural content and native folding via red shift (tryptophan fluorescence). The single chain fragments have shown good binding with HIV-1 gp120 thus validating the expression and refolding strategy and also reinstating E.coli as model expression system for recombinant antibody engineering. SPR based binding analysis coupled with E.coli based expression and purification will have implication for HIV therapeutics and will set a benchmark for future studies of similar kind.

Keywords: Affinity purification; Bacterial expression system; Immunoglobulins; Recombinant antibody; Refolding; SPR.

Figures

Fig. 1
Fig. 1
Far-UV CD spectrum of b12ScFv and b12Scfab. The spectrum was buffer corrected and was obtained at 25 °C with 10 μM of protein in 10 mM Tris HCl buffer, pH 7.4 with a 0.1 cm path length cuvette, a scan-rate of 50 nm/min, a response time of 4 s and a bandwidth of 2 nm. Data reported are averaged over 3 scans.
Fig. 2
Fig. 2
Fluorescence emission spectrum of native and unfolded b12ScFv (left) and b12ScFab (right). The spectrum was obtained at 25 °C with a final protein concentration of 5 μM in 10 mM Tris HCl buffer, pH 7.4 (dashed line) or in presence of 8 M Guanidine Hydrochloride in 10 mM Tris HCl buffer, pH 7.4 (solid line). Each spectrum was buffer corrected. The excitation was at 280 nm and emission was recorded from 300 to 400 nm.
Fig. 3
Fig. 3
Biacore 2000 Sensorgram overlays for the binding kinetics of different concentrations of refolded b12 ScFv to surface-immobilized HIV-1 gp120. Curves 1, 2, 3, 4, and 5 indicate 245, 375, 496, 744, and 1860 nM concentrations of b12 ScFv, respectively. Surface density: 1000 resonance units; buffer: 10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% P20; flow rate: 30 µl/min; temperature: 298 K.
Fig. 4
Fig. 4
Biacore 2000 Sensorgram overlays for the binding kinetics of different concentrations of refolded b12 ScFab to surface-immobilized HIV-1 gp120. Curves 1, 2, 3, 4, and 5 indicate 245, 375, 496, 744, and 1860 nM concentrations of b12 ScFv, respectively. Surface density: 1000 resonance units; buffer: 10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% P20; flow rate: 30 µl/min; temperature: 298 K.

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