Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase. METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration. RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion. CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.

Development of an in vitro aggregation assay for long synthetic polypeptide, amyloidogenic gelsolin fragment AGelD187N 173-242.

Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173-242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173-242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173-242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.

Selection of biophysically favorable antibody variants using a modified Flp-In CHO mammalian display platform.

Mammalian display enables the selection of biophysically favorable antibodies from a large IgG antibody library displayed on the plasma membrane of mammalian cells. We constructed and validated a novel mammalian display platform utilizing the commercially available Flp-In CHO cell line as a starting point. We introduced a single copy of a landing pad for Bxb1 integrase-driven recombinase-mediated cassette exchange into the FRT site of the Flp-In CHO line to facilitate the efficient single-copy integration of an antibody display cassette into the genome of the cell line. We then proceeded to demonstrate the ability of our platform to select biophysically favorable antibodies from a library of 1 × 106 displayed antibodies designed to improve the biophysical properties of bococizumab via randomization of problematic hydrophobic surface residues of the antibody. Enrichment of bococizumab variants via fluorescence-activated cell sorting selections was followed by next generation sequencing and thorough characterization of biophysical properties of 10 bococizumab variants that subsequently allowed attribution of the mutations to the biophysical properties of the antibody variants. The mammalian displayed variants exhibited reduced aggregation propensity and polyreactivity, while critically retaining its target binding thereby demonstrating the utility of this valuable tool.

Optimized Workflow for Selecting Peptides for HDX-MS Data Analyses.

Hydrogen deuterium exchange measured by mass spectrometry (HDX-MS) is a commonly used technique for studying the structural dynamics of proteins in solution. The first part of any bottom-up HDX-MS experiment is to identify the peptides generated from a digestion step. This requires manual inspection of the identified peptides to determine their use for HDX-MS analysis, which is a time-consuming task. Throughout the literature, there have been different approaches for removing peptides that do not yield quantifiable HDX information. This includes using validity scores from the software used in the generation of the peptide map and that the peptide should be found in two out of three technical replicate experiments. Here, we analyze the previously available methods for filtering the identified peptides in regard to their ability to predict whether a peptide will provide quantifiable HDX-MS data or not. We also present a new score-based system relying on a combination of MS/MS parameters that offers an improved method for separating quantifiable peptides from the nonquantifiable. Using this score-based method reduces the number of peptide spectra that needs to be manually inspected and thereby the time spent curating HDX-MS data. Graphical Abstract ᅟ.

Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry.

Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as its reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions.

The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance.

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies.

Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers.

RATIONALE: The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values. METHODS: Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1. RESULTS: Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS: Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2).

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.