Protein Binder for Affinity Purification of Human Immunoglobulin Antibodies

Abstract

The importance of a downstream process for the purification of immunoglobulin antibodies is increasing with the growing application of monoclonal antibodies in many different areas. Although protein A is most commonly used for the affinity purification of antibodies, certain properties could be further improved: higher stability in alkaline solution and milder elution condition. Herein, we present the development of Fc-specific repebody by modular engineering approach and its potential as an affinity ligand for purification of human immunoglobulin antibodies. We previously developed the repebody scaffold composed of Leucine-rich repeat (LRR) modules. The scaffold was shown to be highly stable over a wide range of pH and temperature, exhibiting a modular architecture. We first selected a repebody that binds the Fc fragment of human immunoglobulin G (IgG) through a phage display and increased its binding affinity up to 1.9 × 10(-7) M in a module-by-module approach. The utility of the Fc-specific repebody was demonstrated by the performance of an immobilized repebody in affinity purification of antibodies from a mammalian cell-cultured medium. Bound-antibodies on an immobilized repebody were shown to be eluted at pH 4.0 with high purity (>94.6%) and recovery yield (>95.7%). The immobilized repebody allowed a repetitive purification process more than ten times without any loss of binding capability. The repebody remained almost intact even after incubation with 0.5 M NaOH for 15 days. The present approach could be effectively used for developing a repeat module-based binder for other target molecules for affinity purification.

Characterization of the binding of shikonin to human immunoglobulin using scanning electron microscope, molecular modeling and multi-spectroscopic methods

“…On the other hand, the framework region (FR) containing FR1, FR2, FR3 and FR4 can provide a supporting framework for CDR of Fab [3,34]. The above information is inferred that HIg probably has more potential binding sites for antigens, semi-antigens or other small molecule drugs, which has been proven by recent studies [46][47][48]. Based on the above discussion, we assume that there is at least one binding site and it not be excluded the possibility of two binding sites.…”

Computer-guided Binding Mode Identification and Affinity Improvement of an LRR Protein Binder without Structure Determination

“…In an effort to exploit the structural and functional features of LRR proteins for biotechnological and medical applications, we previously developed an LRR protein binder, called “repebody (Rb)” (27). As a proof of concept, in this work, a human IgG 1 (hIgG 1 )-binding repebody (RbF4) (28) was targeted for the binding mode prediction and affinity maturation without the structure determination. There is indirect evidence that RbF4 binds to the constant region of hIgG 1 (hFc) (28, 29), but actual epitopes and the binding mode were completely unknown.…”

“…As a proof of concept, in this work, a human IgG 1 (hIgG 1 )-binding repebody (RbF4) (28) was targeted for the binding mode prediction and affinity maturation without the structure determination. There is indirect evidence that RbF4 binds to the constant region of hIgG 1 (hFc) (28, 29), but actual epitopes and the binding mode were completely unknown. RbF4 has a typical LRR protein sequence motif, whose structural scaffold consists of three major parts: 1) an N terminal cap (LRRNT), 2) LRR modules (LRRVs), and 3) a C terminal cap (LRRCT) with an additional loop ( Fig.…”

Repeat Module‐Based Rational Design of a Photoswitchable Protein for Light‐Driven Control of Biological Processes

“…To assess the utility of our design strategy,weintroduced cysteine mutations of several pairs into ahuman IgG-specific repebody (D10), which was previously developed. [10] We selected two C b pairs,H 76C/N102C and H76C/G103C,a s typical candidates for the diagonal conjugation of BSBCA.In addition, Q122C/K150C and Q146C/E174C were selected as equivalent pairs to H76C/N102C.F or comparison, D77C/ S123C was also selected for the perpendicular conjugation of BSBCA.V ariants of five C b pairs were constructed using D10, followed by the conjugation of BSBCA.W ep urified the resulting variants,a nd checked the conjugation efficiencyo f BSBCA ( Figure S3). All of the variants revealed asingle peak shift in their mass spectra corresponding to a1 :1 ratio between BSBCA and ar epebody.T his result implies ah igh cross-linking possibility of BSBCA with two cysteine residues in each variant.…”

“…[1,2,[5][6][7] We previously developed ap rotein scaffold, termed repebody,c omposed of LRR (Leucine-rich repeat) modules. [8] Like other LRR proteins,t he repebody comprises ac oncave face of a b-sheet and ac onvex side of the helix array,w hich are responsible for recognizing ac ognate target and maintaining the solenoid structure,respectively,showing high physicochemical stability [8][9][10] (Scheme 1a). Herein, we present ag eneralized strategy to design ap hotoswitchable protein composed of LRR modules for light-driven control of biological processes.T he repebody and an azobenzene derivative,BSBCA (3,3'-bis (sulfonate)-4,4'-bis (chloroacetamido), were used as am odel protein scaffold and ap hotochromic ligand, respectively (Scheme 1b).…”