Activity fingerprinting of AMR ß-lactamase towards a fast and accurate diagnosis.

Antibiotic resistance has become a serious threat to global public health and economic development. Rapid and accurate identification of a patient status for antimicrobial resistance (AMR) are urgently needed in clinical diagnosis. Here we describe the development of an assay method for activity fingerprinting of AMR ß-lactamases using panels of 7 ß-lactam antibiotics in 35 min. New Deli Metallo ß-lactamase-1 (NDM-1) and penicillinase were demonstrated as two different classes of ß-lactamases. The panel consisted of three classes of antibiotics, including: penicillins (penicillin G, piperacillin), cephalosporins (cefepime, ceftriaxone, cefazolin) and carbapenems (meropenem and imipenem). The assay employed a scheme combines the catalytic reaction of AMR ß-lactamases on antibiotic substrates with a flow-injected thermometric biosensor that allows the direct detection of the heat generated from the enzymatic catalysis, and eliminates the need for custom substrates and multiple detection schemes. In order to differentiate classes of ß-lactamases, characterization of the enzyme activity under different catalytic condition, such as, buffer composition, ion strength and pH were investigated. This assay could provide a tool for fast diagnosis of patient AMR status which makes possible for the future accurate treatment with selected antibiotics.

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant.

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

Biocontrol Effect of Clonostachys rosea on Fusarium graminearum Infection and Mycotoxin Detoxification in Oat (Avena sativa).

Oat (Avena sativa) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum. The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea-inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea-treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea-treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.

Conformational Dynamics of Phytoglobin BvPgb1.2 from Beta vulgaris ssp. vulgaris.

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins' roles in planta.

Calorimetric Characterisation of the Binding Reaction Between Human Ferric Haemoglobins and Haptoglobin to Develop a Drug for Removal of Cell-Free Haemoglobin.

High levels of cell-free haemoglobin (Hb) may occur in plasma as a consequence of e.g., pathological haemolysis or blood transfusion. These Hb molecules can be removed from blood circulation by forming a complex with the acute-phase protein haptoglobin (Hp) and thereby can also the intrinsic toxicity of free Hb be limited. In this study it is shown that ferric HbA, HbF, HbE and HbS, respectively, all bind firmly to Hp at 25 °C. By using isothermal titration calorimetry (ITC), it is demonstrated that ferric HbF has higher affinity to Hp (Ka = 2.79 ± 0.29 ×109 M-1) compared with HbA and HbS (1.91 ± 0.24 ×109 M-1) and 1.41 ± 0.34 ×109 M-1 for HbA and HbS, respectively. In addition, the affinity constant for HbE is slightly lower than the other haemoglobins (0.47 ± 0.40 ×109 M-1). Since Hp shows a general and high affinity to all Hb variants tested, it can be concluded that Hp may be useful as a therapeutic agent for several different haemolytic conditions by intravenous injection.

Interaction of haemin with albumin-based macroporous cryogel: Adsorption isotherm and fluorescence quenching studies.

Albumin-based cryogels for capturing haemin were synthesised by crosslinking different biomolecules, bovine serum albumin (BSA) and ovalbumin (OVA). The impact of the protein and coupling agent concentrations on cryogel's mechanical properties, swelling ratios and polymerisation yields, as well as autoclaving as a post-treatment on the cryogel, were studied. We found that BSA (50 mg/ml) and the crosslinker (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 46 mg/ml) formed a cryogel with optimum physical characteristics at a comparatively low protein concentration. The cryogel's mechanical stability was increased using a double-layer cryogel approach by crosslinking the BSA proteins at subzero temperature inside an acrylamide and hydroxyethyl methacrylate premade cryogels. Batch binding and kinetic adsorption isotherms of haemin on the cryogels were assessed to evaluate their binding capacity toward the porphyrin molecule. The results showed that single-layer cryogels (BSA and OVA) had a higher capacity (∼0.68 mg/ml gel) and higher reaction rate constant towards haemin adsorption than double-layer gels. In contrast, the double-layer cryogels had higher mechanical strength than single-layer gels. The experimental results suggested that the cryogels followed the Freundlich model and the pseudo-second-order isotherm for batch adsorption and kinetics, respectively. The interaction between haemin and the gels was studied by fluorescence quenching. We found between 1.1 and 1.6 binding sites for different cryogels.

Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2.

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure.

Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification.

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

Rapid Separation of Human Hemoglobin on a Large Scale From Non-clarified Bacterial Cell Homogenates Using Molecularly Imprinted Composite Cryogels.

The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions.

Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli.

Fetal hemoglobin (HbF) has been developed into an important alternative protein for oxygen therapeutics. Such applications require extensive amounts of proteins, which only can be achieved via recombinant means. However, the expression of vertebrate hemoglobins in heterologous hosts is far from trivial. There are several issues that need to be dealt with. These include, among others, the solubility of the globin chains, equimolar expression of the globin chains, and access to high levels of free heme. In this study, we examined the impact of introducing negative charges on the surface of HbF. Three different HbF mutants were examined, carrying four additional negative charges on the α-subunit (rHbFα4), two additional negative charges on the γ-subunit (rHbFγ2) or a combination of these (rHbFα4/γ2). The increase in negative surface charge in these HbF mutants required the development of an alternate initial capture step in the downstream purification procedures. For the rHbFα4 mutant, we achieved a significantly enhanced yield of purified HbF with no apparent adverse effects on Hb functionality. However, the presence of non-functional Hb portions in the rHbFγ2 and rHbFα4/γ2 samples reduced the yields significantly for those mutants and indicated an imbalanced expression/association of globin chains. Furthermore, the autoxidation studies indicated that the rHbFγ2 and rHbFα4/γ2 mutants also were less oxidatively stable than rHbFα4 and wt rHbF. The study further verified the need for an improved flask culture protocol by optimizing cultivation parameters to enable yield-improving qualities of surface-located mutations.

Rapid personalized AMR diagnostics using two-dimensional antibiotic resistance profiling strategy employing a thermometric NDM-1 biosensor.

Antimicrobial resistance (AMR) threatens global public health and modern surgical medicine. Expression of ß-lactamase genes is the major mechanism by which pathogens become antibiotic resistant. Pathogens expressing extended spectrum ß-lactamases (ESBL) and carbapenemases (CP) are especially difficult to treat and are associated with increased hospitalization and mortality rates. Despite considerable effort, identification of ESBLs and CPs in a clinically relevant timeframe remains challenging. In this study, a two-dimensional AMR profiling assay strategy was developed employing panels of antibiotics (penicillins, cephamycins, oximino-cephalosporins and carbapenems) and ß-lactamases inhibitors (avibactam and EDTA). The assay required the development of a novel biosensor that employed New Delhi metallo-ß-lactamase-1 (NDM-1) as the sensing element. Functionally probing ß-lactamase activity using substrates and inhibitors combinatorically increased the informational content that enabled the development of assays capable of simultaneous, differential identification of multiple ß-lactamases expressed in a single bacterial isolate. More specifically, the assay enabled the simultaneous identification of ESBL and CP in mock samples, as well as in an engineered construct which co-expressed these ß-lactamases. The NDM-1 biosensor assay was 16 times and 8 times more sensitive than the ESBL Nordmann/Dortet/Poirel (NDP) and Carba Nordmann/Poirel (NP) assays, respectively. In a retrospective study, NDM-1 biosensor assays were able to differentially identify ESBLs, metallo-CPs and serine-CPs ß-lactamases in 23 clinical isolates with 100% accuracy. An assay algorithm was developed which accelerated data analytics reducing turnaround to <1 h. The assay strategy integrated with AI-based data analytics has the potential to provide physicians with a comprehensive readout of patient AMR status.

Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers.

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.

Synthesizing a Hybrid Nanocomposite as an Affinity Adsorbent through Surface-Initiated Atom Transfer Radical Polymerization Catalyzed by Myoglobin.

A hybrid bifunctional core-shell nanostructure was synthesized for the first time via surface-initiated atom transfer radical polymerization (SI-ATRP) using myoglobin as a biocatalyst (ATRPase) in an aqueous solution. N-Isopropyl acrylamide (NIPA) and N-(3-aminopropyl)methacrylamide (APMA) were applied to graft flexible polymer brushes onto initiator-functionalized silica nanoparticles. Two different approaches were implemented to form the core-shell nanocomposite: (a) random copolymerization, Si@p(NIPA-co-APMA) and (b) sequential block copolymerization, Si@pNIPA-b-pAPMA. These nanocomposites can be used as versatile intermediates, thereby leading to different types of materials for targeted applications. In this work, a phenylboronic acid ligand was immobilized on the side chain of the grafted brushes during a series of postmodification reactions to create a boronate affinity adsorbent. The ability to selectively bind glycoproteins (ovalbumin and glycated hemoglobin) via boronic acid was assessed at two different temperatures (20 and 40 °C), where Si@pNIPA-b-APMABA (163 mg OVA/g of particle) displayed an approximately 1.5-fold higher capacity than Si@p(NIPA-co-APMA)BA (107 mg OVA/g of particle). In addition to selective binding to glycoproteins, the nanocomposites exhibited selective binding for myoglobin due to the molecular imprinting effect during the postmodification process, that is, 72 and 111 mg Mb/g for Si@p(NIPA-co-APMA)BA and Si@pNIPA-b-pAPMABA, respectively.

Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin.

Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb.

Expression, Purification and Initial Characterization of Functional α1-Microglobulin (A1M) in Nicotiana benthamiana.

α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.

Multimeric fusion single-chain variable fragments as potential novel high-capacity ligands.

In basic and applied biotechnology, design of affinity ligands has become essential for high-capacity applications such as affinity-based downstream processes for therapeutic molecules. Here, we established a proof-of-concept for the use of multimeric fusion single-chain variable fragment (scFvs) as high-capacity ligands in affinity adsorbents. Mono- and di/tri-scFvs separated by Pro-rich negatively charged linkers were designed, produced, and immobilized to 6% cross-linked agarose beads. Frontal binding experiments with a target protein of 50 kDa resulted in up to 20 mg·mL-1 and 82% in dynamic binding capacity and utilization yield, respectively, at 100% breakthrough. The utilization of the binding sites was impacted by the ligand format and ligand density, rather than limitation in pore size of adsorbent as previously suggested. Overall, we demonstrated that multimeric fusion scFvs can successfully be developed and used as high-capacity ligands in affinity adsorbents, enabling lean process design and alignment with process specifications.

Plant based production of myoglobin - a novel source of the muscle heme-protein.

Myoglobin is a heme-protein in the muscle of vertebrates with important functions in the oxygenation of tissues and as a regulator in nitric oxide signaling. Myoglobin from many species is also an important nutritional source of bioavailable iron. In this study, we have successfully produced human myoglobin in the leaves of Nicotiana benthamiana by transient expression using a viral vector delivered by Agrobacterium tumefaciens. Analyses confirmed that heme was incorporated and the protein was functional, with observed properties consistent with those of native myoglobins. A relatively high degree of purity could be achieved with low cost methods. The results show the high potential of plants as a production platform for heme proteins, a group of proteins of interest for iron nutrition applications and possible future pharmaceutical development.

Sugar beet hemoglobins: reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism.

In contrast with human hemoglobin (Hb) in red blood cells, plant Hbs do not transport oxygen, instead research points towards nitrogen metabolism. Using comprehensive and integrated biophysical methods we characterized three sugar beet Hbs: BvHb1.1, BvHb1.2 and BvHb2. Their affinities for oxygen, CO, and hexacoordination were determined. Their role in nitrogen metabolism was studied by assessing their ability to bind NO, to reduce nitrite (NiR, nitrite reductase), and to form nitrate (NOD, NO dioxygenase). Results show that BvHb1.2 has high NOD-like activity, in agreement with the high nitrate levels found in seeds where this protein is expressed. BvHb1.1, on the other side, is equally capable to bind NO as to form nitrate, its main role would be to protect chloroplasts from the deleterious effects of NO. Finally, the ubiquitous, reactive, and versatile BvHb2, able to adopt 'open and closed forms', would be part of metabolic pathways where the balance between oxygen and NO is essential. For all proteins, the NiR activity is relevant only when nitrite is present at high concentrations and both NO and oxygen are absent. The three proteins have distinct intrinsic capabilities to react with NO, oxygen and nitrite; however, it is their concentration which will determine the BvHbs' activity.

Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions.

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.

Chromatographic separation of hemoglobin variants using robust molecularly imprinted polymers.

Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mL-1resin for adult Hb (HbA) and fetal Hb (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA αY42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an αA19D-αA12D HbF mutant eluted earlier, at -2.7 column volumes compared to wildtype HbF.