AQP4-DARPin1: A Chimeric Antigen Based on Scaffold Protein DARPin for Efficient Detection of AQP4-IgG in NMOSD.

AQP4-IgG is an autoantibody associated with neuromyelitis optica spectroscopic disorder (NMOSD), a central nervous system inflammatory disease that requires early diagnosis and treatment. We designed two fusion proteins, AQP4-DARPin1 and AQP4-DARPin2, comprising the complete antigenic epitopes of aquaporin-4 (AQP4) and the constant region of the scaffold protein DARPin. These fusion proteins were expressed and purified from Escherichia coli and coated on microplates to develop an efficient method for detecting AQP4-IgG. Molecular dynamics simulation revealed that the fusion of AQP4 extracellular epitopes with DARPin did not alter the main structure of DARPin. The purified AQP4-DARPins bound recombinant antibody rAb-53 (AQP4-IgG) with affinities of 135 and 285 nM, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation demonstrated that AQP4-DARPin1 specifically recognized AQP4-IgG in the NMOSD patient serum. AQP4-DARPin1 as a coated antigen showed higher ELISA signal and end point dilution ratio than full-length AQP4. Our AQP4-DARPin1-coated AQP4-IgG ELISA had 100% specificity and 90% sensitivity. These results indicate that AQP4-DARPin1, compared to existing detection strategies that use full-length or extracellular loop peptides of AQP4, provides a new and more effective approach to the ELISA detection of NMOSD.

Reduction-Activatable Fluorogenic Nanobody for Targeted and Low-Background Bioimaging.

Environment-sensitive fluorogenic antibodies enable target-specific bioimaging with reduced unspecific background signal and improved spatiotemporal resolution. However, current strategies for the construction of fluorogenic antibodies are hard to handle due to challenges that lie in the prior design of fluorogenic probes and subsequent antibody labeling. Here, we report a simple strategy to generate a fluorogenic nanobody, which we term D-body, by in situ incorporation of a reduction-responsive Nile blue foldamer which is self-quenched via a dimerization-caused quenching mechanism. The D-body can be efficiently internalized by cells with high epidermal growth factor receptor expression levels and is highly fluorogenic upon lysosomal activation, allowing wash-free cell imaging with exquisite specificity and fast in vivo imaging with a high tumor-to-background ratio. The modular D-body is readily available and easy to handle, offering a platform that is highly tunable for bioimaging applications.

One-Step Assembly of Versatile Multifunctional Coatings Based on Host-Guest and Polyphenol Chemistry.

Developing a facile, efficient, and versatile polyphenol coating strategy and exploring its novel applications are of great significance in the fields of material surfaces and interfaces. Herein, a one-step assembly strategy for constructing novel tannic acid (TA) coatings via a solvent evaporation method is reported using TA and polycyclodextrin (PCD) particles (TPP). TPP with a high phenolic group activity of 88% integrates the advantages of host-guest and polyphenol chemistry. The former can drive TPP dynamically assemble into a large and collective aggregation activated by high temperature or density, and the latter provides excellent adhesion properties to substrates (0.9 mg cm-2 ). TPP can assemble into a coating (TPC) rapidly on various substrates within 1 h at 37 °C while with a high availability of feed TPP (≈90%). The resulting TPC is not only high-temperature steam-sensitive for use as an anti-fake mask but also pH-sensitive for transforming into a free-standing film under physiological conditions. Moreover, various metal ions and functional particles can incorporate into TPC to extend its versatile properties including antibacterial activity, enhanced stability, and conductivity. This work expands the polyphenol coating strategy and builds up a one-step and efficient preparation platform of polyphenol coating for multiapplication prospects in various fields.

SnoopLigase Enables Highly Efficient Generation of C-C-Linked Bispecific Nanobodies Targeting TNF-α and IL-17A.

Bispecific antibodies (bis-Nbs) have been extensively developed since the concept was devised over the decades. Taking advantage of the superior characteristics of nanobodies, bis-Nbs exhibit an emerging tendency to become the new generation of research and diagnostic tools. Traditional strategies to connect the homo- or heterogeneous monomers are commonly applied, but there are still technical issues to generate the bispecific molecules as efficiently as designed. Here, we utilize SnoopLigase to directly tether the C terminus (C-C) of the tagged nanobodies against tumor necrosis factor-α (TNF-α) and interleukin-17A (IL-17A). Under optimal conditions, the yield of C-C-linked bis-Nbs can reach as high as 70% due to the existence of SnoopLigase. The prepared bis-Nbs possessed similar or even higher affinity as the monomers and significantly inhibited the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) induced by TNF-α and IL-17A. This study provides an innovative route for using SnoopLigase to realize a highly efficient generation of C-C-linked bis-Nbs. The approach can be applied to different and multicomponent systems for their potential applications in disease diagnosis and treatment.

Tyrosine-Based Dual-Functional Interface for Trapping and On-Site Photo-Induced Covalent Immobilization of Proteins.

Tyrosine, a simple and well-available natural amino acid, is featured by the small size of the compound that contains multiple reactive groups. This study developed an efficient bioconjugation strategy using tyrosine-based dual-functional interfaces. When tyrosine molecules are immobilized on the surface of a supporting material through amino groups, their carboxyl groups can function as an attracting trap due to their anionic nature at neutral pH and ability to chelate nickel(II) ions (Ni2+), allowing the capture and enrichment of cationic proteins and histidine (His)-tagged proteins on the surface. The trapped proteins can be further covalently immobilized on site through ruthenium-mediated photochemical cross-linking, which has been found to be highly efficient and can be completed within minutes. This strategy was successfully applied to two different material systems. We found that tyrosine-modified agarose beads had a binding capacity of the His-tagged enhanced green fluorescent protein comparable to that of commonly used nitrilotriacetic acid-based resins, and further covalent coupling via dityrosine cross-linking achieved a yield of 85% within 5 min, without compromising much on its fluorescence activity. On the surface of tyrosine-modified 316L stainless steel, lysozyme was captured through electrostatic interaction and further immobilized. The resultant surface exhibited remarkable antibacterial activity against both Staphylococcus aureus and Escherichia coli. Such a tyrosine-based capture-then-coupling method is featured by its simplicity, high coupling efficiency, and high utilization rate of target molecules, making it particularly suitable for the proteins that are highly priced or vulnerable to general immobilization chemistry.

Freezing-assisted synthesis of covalent C-C linked bivalent and bispecific nanobodies.

Bi-valent/specific antibodies are coming to the forefront of therapeutic and diagnostic applications for extending the functions of conventional antibodies. Nanobodies as building blocks, due to their small sizes, are prone to synthesizing these homo/hetero-dimers. However, the classical C-terminus to N-terminus (C-N) ligation manner for generating the dimer results in the inhibition of the antigen-binding capacity of the bivalent/specific antibodies. In this study, we designed and constructed several C-terminus to C-terminus (C-C) linked bivalent and bispecific nanobodies against the human ß2-microglobulin via freezing, overcoming the biological function-disrupt raised by the C-N ligation. The nanobody modified by the formylglycine generating enzyme was ligated to a hydrazide or aminooxy bi-functionalized linker. During the process, we discovered that freezing significantly improved the efficiency of hydrazone or oxime formation between the linker and nanobodies, which could not take place at room temperature. By freezing from -10 to -20 °C, up to 50% yield of bivalent nanobodies was achieved within 24 h. The C-C linked nanobody-fusions maintained almost all of its binding activity and exhibited an increase by two orders of magnitudes in affinity kinetics, demonstrating the superiority of C-C over the C-N linking approach.

Generation and Application of Fluorescent Anti-Human ß2-Microglobulin VHHs via Amino Modification.

The functionalization of VHHs enables their application in almost every aspect of biomedical inquiry. Amino modification remains a common strategy for protein functionalization, though is considered to be inferior to site-specific methods and cause protein property changes. In this paper, four anti-ß2M VHHs were selected and modified on the amino group by NHS-Fluo. The impacts of amino modification on these VHHs were drastically different, and among all th examples, the modified NB-1 maintained the original stability, bioactivity and homogeneity of unmodified NB-1. Specific recognition of VHHs targeting ß2M detected by fluorescence imaging explored the possible applications of VHHs. Via this study, we successfully functionalized the anti-ß2M VHHs through amino modification and the results are able to instruct the simple and fast functionalization of VHHs in biomedical researches.

High Expression Achievement of Active and Robust Anti-ß2 microglobulin Nanobodies via E.coli Hosts Selection.

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-ß2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-ß2 microglobulin (ß2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10-8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-ß2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124.

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10-90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (eep 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

Structural and enzymatic characterization of acetolactate decarboxylase from Bacillus subtilis.

Acetoin is an important physiological metabolite excreted by microbes. Its functions include avoiding acidification, participating in regulation of the NAD+/NADH ratio, and storing carbon. Acetolactate decarboxylase is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin). It catalyzes conversion of the (R)- and (S)-enantiomers of acetolactate to generate the single product, (R)-acetoin. In addition to the X-ray crystal structure of acetolactate decarboxylase from Bacillus brevis, although the enzyme is widely present in microorganisms, very few atomic structures of acetolactate decarboxylase are reported. In this paper, we solved and reported a 1.5 Å resolution crystal structure of acetolactate decarboxylase from Bacillus subtilis. Dimeric assembly is observed in the solved structure, which is consistent with the elution profile conducted by molecular filtration. A zinc ion is coordinated by highly conserved histidines (191, 193, and 204) and conserved glutamates (62 and 251). We performed kinetic studies on acetolactate decarboxylase from Bacillus subtilis using circular dichroism, allowing the conversion of acetolactate to chiral acetoin for real-time tracking, yielding a Km value of 21 mM and a kcat value of 2.2 s-1. Using the two enantiomers of acetolactate as substrates, we further investigated the substrate preference of acetolactate decarboxylase from Bacillus subtilis by means of molecular docking and dynamic simulation in silico. The binding free energy of (S)-acetolactate was found to be ~ 30 kcal/mol greater than that of (R)-acetolactate, indicating a more stable binding for (S)-acetolactate.

Characterization of a salt-activated protease with temperature-dependent secretion in Stenotrophomonas maltophilia FF11 isolated from frozen Antarctic krill.

Seafood is sometimes wasted due to the growth of psychrotolerant microbes which secrete proteases and break down proteins. Stenotrophomonas maltophilia FF11, isolated from frozen Antarctic krill, grows at a wide range of temperatures and secretes more proteases at low temperatures. According to zymogram analysis, two kinds of proteases were produced from this strain. A major protease was produced largely at 15 °C, but not at 37 °C. The temperature-dependent secreted protease was purified to homogeneity. Its molecular mass was determined at 37.4 kDa and its amino acid sequence was also obtained. This protease is a member of the subtilase group according to the NCBI blast analysis. The enzyme was highly stable at high salt concentration (4 M). Interestingly, its activity increased about 1.6-fold under high salt condition. The enzyme remains active and stable in different organic solvents (50 %, v/v) such as dimethylsulfoxide, dimethyl formamide, dioxane and acetone. These properties may provide potential applications in quality control for sea foods, in protein degradation at high salt concentration, in biocatalysis and biotransformation within non-aqueous media, such as detergent and transesterification.

Deactivation of Mcl-1 by Dual-Function Small-Molecule Inhibitors Targeting the Bcl-2 Homology 3 Domain and Facilitating Mcl-1 Ubiquitination.

By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. NoxaBH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, BimBH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.

The human W42R γD-crystallin mutant structure provides a link between congenital and age-related cataracts.

Some mutants of human γD-crystallin are closely linked to congenital cataracts, although the detailed molecular mechanisms of mutant-associated cataract formation are generally not known. Here we report on a recently discovered γD-crystallin mutant (W42R) that has been linked to autosomal dominant, congenital cataracts in a Chinese family. The mutant protein is much less soluble and stable than wild-type γD-crystallin. We solved the crystal structure of W42R at 1.7 Šresolution, which revealed only minor differences from the wild-type structure. Interestingly, the W42R variant is highly susceptible to protease digestion, suggesting the presence of a small population of partially unfolded protein. This partially unfolded species was confirmed and quantified by NMR spectroscopy. Hydrogen/deuterium exchange experiments revealed chemical exchange between the folded and unfolded species. Exposure of wild-type γD-crystallin to UV caused damage to the N-terminal domain of the protein, resulting in very similar proteolytic susceptibility as observed for the W42R mutant. Altogether, our combined data allowed us to propose a model for W42R pathogenesis, with the W42R mutant serving as a mimic for photodamaged γD-crystallin involved in age-related cataract.

Chimeric AQP4-based immunosorbent for highly-specific removal of AQP4-IgG from blood.

Anti-aquaporin-4 autoantibodies (AQP4-IgG) are implicated in the pathogenesis of neuromyelitis optica spectrum disorders (NMOSD), and their removal from the blood circulation is considered to be an effective method for acute treatment. An ideal extracorporeal AQP4-IgG removal system should have high specificity, which means that it can selectively remove AQP4-IgG without affecting normal immunoglobulins. However, the conventional tryptophan immobilized column lacks sufficient specificity and cannot achieve this goal. In this study, we successfully prepared a fusion protein chimeric AQP4, which consists of the complete antigenic epitopes of human AQP4 and the constant region of scaffold protein DARPin. Chimeric AQP4 was expressed and purified from Escherichia coli, and then immobilized on agarose gel as a ligand for selective capture of AQP4-IgG immunosorbent. The prepared immunosorbent had a theoretical maximum adsorption capacity of 20.48 mg/g gel estimated by Langmuir isotherm. In vitro plasma perfusion tests demonstrated that the chimeric AQP4 coupled adsorbent had remarkable adsorption performance, and could eliminate more than 85 % of AQP4-IgG under the gel-to-plasma ratio of 1:50. Moreover, it exhibited high specificity because other human plasma proteins were not adsorbed in the dynamic adsorption experiment. These results suggest that the chimeric AQP4 coupled immunosorbent can provide a new approach for specific immunoadsorption (IA) treatment of NMOSD.

Crystal structure of the cataract-causing P23T γD-crystallin mutant.

Up to now, efforts to crystallize the cataract-associated P23T mutant of human γD-crystallin have not been successful. Therefore, insights into the light scattering mechanism of this mutant have been exclusively obtained from solution work. Here we present the first crystal structure of the P23T mutant at 2.5 Šresolution. The protein exhibits essentially the same overall structure as seen for the wild-type protein. Based on our structural data, we confirm that no major conformational changes are caused by the mutation, and that solution phase properties of the mutant appear exclusively associated with cataract formation.

Structural and enzymatic characterization of Bacillus subtilis R,R-2,3-butanediol dehydrogenase.

2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.

Nanobody-functionalized conduit with built-in static mixer for specific elimination of cytokines in hemoperfusion.

Removing excessively produced cytokines is of paramount significance in blood purification therapy for hypercytokinemia-associated diseases. In this study, we devised a conduit that is modified with nanobodies (Nb) and incorporates static mixers (Nb-SMC) to eliminate surplus cytokines from the bloodstream. The low-pressure-drop (LPD) static mixer, with each unit featuring two 90°-crossed blades, was strategically arranged in a tessellated pattern on the inner wall of the conduit to induce turbulent mixing effects during the flow of blood. This arrangement enhances mass transfer and molecular diffusion, thereby assisting in the identification and elimination of cytokines. By utilizing computational fluid dynamics (CFD) studies, the Nb-SMC was rationally designed and prepared, ensuring an optimal interval between two mixer units (H/G = 2.5). The resulting Nb-SMC exhibited a remarkable selective clearance of IL-17A, reaching up to 85 %. Additionally, the process of Nb immobilization could be adjusted to achieve the simultaneous removal of multiple cytokines from the bloodstream. Notably, our Nb-SMC displayed good blood compatibility without potential adverse effects on the composition of human blood. As the sole documented static mixer-integrated conduit capable of selectively eliminating cytokines at their physiological concentrations, it holds promise in the clinical potential for hypercytokinemia in high-risk patients. STATEMENT OF SIGNIFICANCE: High-efficient cytokines removal in critical care still remains a challenge. The conduit technique we proposed here is a brand-new strategy for cytokines removal in blood purification therapy. On the one hand, nanobody endows the conduit with specific recognition of cytokine, on the other hand, the build-in static mixer enhances the diffusion of antigenic cytokine to the ligand. The combination of these two has jointly achieved the efficient and specific removal of cytokine. This innovative material is the only reported artificial biomaterial capable of selectively eliminating multiple cytokines under conditions close to clinical practice. It has the potential to improve outcomes for patients with hypercytokinemia and reduce the risk of adverse events associated with current treatment modalities.

Facile surface treatment strategy to generate dense lysozyme layer on ultra-high molecular weight polyethylene enabling inhibition of bacterial biofilm formation.

Medical plastics such as those found in endotracheal tubes are widely used in intensive care units for the treatment of critically ill patients. Although commonplace in hospital environment, these catheters are at a high risk of bacterial contamination and have been found responsible for numerous health-care-associated infections. Antimicrobial coatings that can prevent harmful bacterial growth are required to reduce the occurrence of such infections. In this study, we introduce a facile surface treatment strategy that could form antimicrobial coatings on the surface of average medical plastics. The strategy involves treatment of activated surfaces with lysozyme, a natural antimicrobial enzyme presenting in human lacrimal gland secretions which is widely used for wound healing. Using ultra-high molecular weight polyethylene (UHMWPE) as the representative surface, oxygen/argon plasma treatment for 3 min led to the increase of surface roughness and the generation of negatively charged groups, with the zeta potential measured as -94.5 mV at pH 7. The activated surface could accommodate lysozyme with a density of up to 0.3 nmol/cm2 through electrostatic interaction. Antimicrobial activity of the resulting surface (UHMWPE@Lyz) was characterized with Escherichia coli and Pseudomonas sp. strains, and the treated surface significantly inhibited the bacterial colonization and the formation of biofilm compared to the untreated UHMWPE. This method of constructing an effective lysozyme-based antimicrobial coating is a generally applicable, simple and fast process for surface treatment with no adverse solvent and wastes involved.

Structural and biochemical characterization of the childhood cataract-associated R76S mutant of human γD-crystallin.

Although a number of γD-crystallin mutations are associated with cataract formation, there is not a clear understanding of the molecular mechanism(s) that lead to this protein deposition disease. As part of our ongoing studies on crystallins, we investigated the recently discovered Arg76 to Ser (R76S) mutation that is correlated with childhood cataract in an Indian family. We expressed the R76S γD-crystallin protein in E. coli, characterized it by CD, fluorescence, and NMR spectroscopy, and determined its stability with respect to thermal and chemical denaturation. Surprisingly, no significant biochemical or biophysical differences were observed between the wild-type protein and the R76S variant, except a lowered pI (6.8 compared to the wild-type value of 7.4). NMR assessment of the R76S γD-crystallin solution structure, by RDCs, and of its motional properties, by relaxation measurements, also revealed a close resemblance to wild-type crystallin. Further, kinetic unfolding/refolding experiments for R76S and wild-type protein showed similar degrees of off-pathway aggregation suppression by αB-crystallin. Overall, our results suggest that neither structural nor stability changes in the protein are responsible for the R76S γD-crystallin variant's association with cataract. However, the change in pI and the associated surface charge or the altered nature of the amino acid could influence interactions with other lens protein species.

An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions.

Camelid-derived nanobody is emerging as a resourceful platform for developing immunoaffinity ligands for chromatography applications. Featured by high affinity and selectivity, BC2 nanobody (BC2-Nb), which can recognize a specific epitope tag (PDRKAAVSHWQQ, termed BC2T), is potential to be developed as a general tool for recombinant protein purification. However, excessively high affinity between binding partners makes the desorption of products less efficient and limits its application. Aiming to improve elution efficiency, structure-guided mutations of BC2-Nb were conducted to adjust the structural flexibility of its antigen-binding site. Six ligand variants were obtained with their binding affinity decreasing by about 100-fold. Among them, one mutated BC2-Nb named 44D was chosen to prepare immunoaffinity resin, and its adsorption and elution performance were well characterized. The site-directed mutation led to the equilibrium dissociation constant (KD) of BC2-Nb changing from 1.4 × 10-9 M to 1.4 × 10-7 M (44D). The resin using 44D as ligand retained a static binding capacity of 19.14 mg/mL toward BC2T-fused enhanced green fluorescent protein (eGFP-BC2T). Significantly improved elution efficiency was obtained with the mutated ligand. Protein recovery reached 94% at pH 3.5 for 44D-based resin, while the resin based on original BC2-Nb could only achieve its highest recovery of 84% at pH 2. In addition, a neutral elution condition (1 M arginine containing 50% propylene glycol, pH 7.4) was also found effective, which allowed a product recovery of 95%. The resin enabled direct capturing of eGFP-BC2T from bacterial lysates, and the one-step purification with the both elution conditions could achieve a product purity of more than 90%. This study provided a promising affinity ligand, and also proved the feasibility of controlling the elution process of nanobody-based affinity resin through the strategy of binding sites modification.