CO2 Hydrogenation to CH3OH on Metal-Doped TiO2(110): Mechanisms, Strain Effect and a New Thermodynamic-Kinetic Relation.

Surface strain and linear thermodynamic-kinetic relation are interesting topics in catalysis. Development of low temperature methanol catalysts of high activity and selectivity is of particularly importance for conversion of CO2 to methanol. In the present paper CO2 hydrogenation to methanol on Znx@TiO2(110) (x=0-2) was explored using density functional calculations and microkinetic simulations. The reaction mechanisms on the three model systems were determined and it is shown that Zn2@TiO2(110) is the most active. The most favorable pathway on Zn2@TiO2(110) is identified and CO2+H to HCOO is found to be the rate-controlling step. It is demonstrated that there is a linear relation (named AEB relation) between the adsorption energies of the initial states and the barriers for the controlling step on the 18 systems studied. Calculations on strained surfaces show that the AEB relation exists within ±1 % strain. Sr2@TiO2(110) and -1 % strained CaZn and ZnCu doped TiO2(110) are potential good low temperature catalysts and deserve experimental testing.

A novel anti-PD-L1/IL-15 immunocytokine overcomes resistance to PD-L1 blockade and elicits potent antitumor immunity.

Despite the demonstrated immense potential of immune checkpoint inhibitors in various types of cancers, only a minority of patients respond to these therapies. Immunocytokines designed to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME) and block the immune checkpoint simultaneously may provide a strategic advantage over the combination of two single agents. To increase the response rate to checkpoint blockade, in this study, we developed a novel immunocytokine (LH01) composed of the antibody against programmed death-ligand 1 (PD-L1) fused to interleukin (IL)-15 receptor alpha-sushi domain/IL-15 complex. We demonstrate that LH01 efficiently binds mouse or human PD-L1 and maintains IL-15 stimulatory activity. In syngeneic mouse models, LH01 showed improved antitumor efficacy and safety versus anti-PD-L1 plus LH02 (Fc-sushi-IL15) combination and overcame resistance to anti-PD-L1 treatment. Mechanistically, the dual anti-immunosuppressive function of LH01 activated both the innate and adaptive immune responses and induced a favorable and immunostimulatory TME. Furthermore, combination therapy with LH01 and bevacizumab exerts synergistic antitumor effects in an HT29 colorectal xenograft model. Collectively, our results provide supporting evidence that fusion of anti-PD-L1 and IL-15 might be a potent strategy to treat patients with cold tumors or resistance to checkpoint blockade.

Oxidation and reduction analysis of therapeutic recombinant human interleukin-15 by HPLC and LC-MS.

Being an important immune stimulant of T lymphocytes and NK cells, the recombinant human interleukin-15 (rhIL-15) has been extensively researched in tumor immunotherapy or as a vaccine adjuvant. However, the rhIL-15 manufacturing level lags far behind its growing clinical demand due to the lack of efficient and exact analysis methodologies to characterize the trace by-products, typically redox and deamidation. In order to improve the production and quality control of rhIL-15, here we developed an expanded resolution reverse-phase high-performance liquid chromatography (ExRP-HPLC) approach to quickly and accurately analyze the oxidation and reduction by-products of rhIL-15, which may appear during the purification processes. Firstly, we developed RP-HPLC methods which can separate rhIL-15 fractions with different levels of oxidization or reduction, respectively, and the redox status of each peak was then determined by measuring the intact mass with a high-resolution mass spectrometer (UPLC-MS). To further clarify the complex pattern of oxidization of specific residues, the peaks with various oxidation levels were digested into pieces for peptide mapping to pinpoint the exact changes of oxygen and hydrogen atoms in the rhIL-15 by-products. In addition, we performed the ExRP-HPLC and UPLC-MS analysis of partially deamidated rhIL-15 to characterize their oxidation and reduction. Our work is the first in-depth characterization of the redox by-products of rhIL-15, even for deamidated impurities. The ExRP-HPLC method we reported can facilitate the rapid and accurate quality analysis of rhIL-15, which is substantially helpful for streamlining the industrial manufacturing of rhIL-15 to better meet the demands of clinical applications. KEYPOINTS: • The oxidization and reduction rhIL-15 by-products were characterized for the first time. • The changes of oxygen and hydrogen atoms in rhIL-15 redox by-products were accurately determined by UPLC-MS. • Oxidation and reduction by-products of deamidated rhIL-15 were further analyzed.

PM2.5-bound polyhalogenated carbazoles (PHCZs) in urban Beijing, China: Occurrence and the source implication.

Polyhalogenated carbazoles (PHCZs) are recently raising much attention due to their toxicity and ubiquitous environmental distribution. However, little knowledge is known about their ambient occurrences and the potential source. In this study, we developed an analytical method based on GC-MS/MS to simultaneously determine 11 PHCZs in PM2.5 from urban Beijing, China. The optimized method provided low method limit of quantifications (MLOQs, 1.45-7.39 fg/m3) and satisfied recoveries (73.4%-109.5%). This method was applied to analyze the PHCZs in the outdoor PM2.5 (n = 46) and fly ash (n = 6) collected from 3 kinds of surrounding incinerator plants (steel plant, medical waste incinerator and domestic waste incinerator). The levels of ∑11PHCZs in PM2.5 ranged from 0.117 to 5.54 pg/m3 (median 1.18 pg/m3). 3-chloro-9H-carbazole (3-CCZ), 3-bromo-9H-carbazole (3-BCZ), and 3,6-dichloro-9H-carbazole (36-CCZ) were the dominant compounds, accounting for 93%. 3-CCZ and 3-BCZ were significantly higher in winter due to the high PM2.5 concentration, while 36-CCZ was higher in spring, which may be related to the resuspending of surface soil. Furthermore, the levels of ∑11PHCZs in fly ash ranged from 338 to 6101 pg/g. 3-CCZ, 3-BCZ and 36-CCZ accounted for 86.0%. The congener profiles of PHCZs between fly ash and PM2.5 were highly similar, indicating that combustion process could be an important source of ambient PHCZs. To the best of our knowledge, this is the first research providing the occurrences of PHCZs in outdoor PM2.5.

SHTXTHHly, an extracellular secretion platform for the preparation of bioactive peptides and proteins in Escherichia coli.

BACKGROUND: In previous work, we developed an E. coli extracellular secretion platform XTHHly based on the hemolysin A secretion system. It can produce bioactive peptides with simple purification procedures. However, the wider application of this platform is limited by poor secretion efficiency. RESULTS: In this study, we first discovered a positive correlation between the isoelectric point (pI) value of the target protein and the secretion level of the XTHHly system. Given the extremely high secretion level of S tag, we fused it at the N-terminus and created a novel SHTXTHHly system. The SHTXTHHly system significantly increased the secretion levels of antimicrobial peptides (PEW300, LL37, and Aurein 1.2) with full bioactivities, suggesting its excellent capacity for secretory production of bioactive peptides. Furthermore, RGDS, IL-15, and alcohol dehydrogenase were successfully secreted, and their bioactivities were largely maintained in the fusion proteins, indicating the potential applications of the novel system for the rapid determination of protein bioactivities. Finally, using the SHTXTHHly system, we produced the monomeric Fc, which showed a high affinity for Fcγ Receptor I and mediated the antibody-dependent immunological effects of immune cells, demonstrating its potential applications in immunotherapies. CONCLUSIONS: The SHTXTHHly system described here facilitates the secretory production of various types of proteins in E. coli. In comparison to previously reported expression systems, our work enlightens an efficient and cost-effective way to evaluate the bioactivities of target proteins or produce them.

DFT and microkinetic study of acetylene transformation on Pd(111), M(111) and PdM(111) surfaces (M = Cu, Ag, Au).

Density functional calculations and microkinetic simulations were performed on the transformation network of acetylene on Pd(111), M(111) and PdM(111) (M = Cu, Ag, Au) surfaces. It is demonstrated that the adsorption energies on alloy surfaces linearly correlate with the values on the pure metal surfaces. A good linear relationship between the co-adsorption energies of initial states and transition states is revealed with which the barriers of most elementary steps in the reaction network were estimated. To shed light on the transformation of acetylene, microkinetic simulations were conducted on the network. The results show that CHCH and H are dominant species on the surfaces and CCH, CCH2 and CCH3 are the main intermediates. Analysis indicates that introduction of coinage metals into Pd reduces the activity, but promotes the selectivity by lowering the barrier of CHCH2 → CH2CH2. The present work provides a comprehensive overview of acetylene transformation on palladium, coinage metals and their alloy surfaces. The linear relationship of adsorption energies between the component metal and alloy surfaces and usage of the TSS relationship to evaluate barriers for microkinetic simulations are worthy of being further studied and extended to other systems.

Anti-bacterial monoclonal antibodies: next generation therapy against superbugs.

Prior to the nineteenth century, infectious disease was one of the leading causes of death. Human life expectancy has roughly doubled over the past century as a result of the development of antibiotics and vaccines. However, the emergence of antibiotic-resistant superbugs brings new challenges. The side effects of broad-spectrum antibiotics, such as causing antimicrobial resistance and destroying the normal flora, often limit their applications. Furthermore, the development of new antibiotics has lagged far behind the emergence and spread of antibiotic resistance. On the other hand, the genome complexity of bacteria makes it difficult to create effective vaccines. Therefore, novel therapeutic agents in supplement to antibiotics and vaccines are urgently needed to improve the treatment of infections. In recent years, monoclonal antibodies (mAbs) have achieved remarkable clinical success in a variety of fields. In the treatment of infectious diseases, mAbs can play functions through multiple mechanisms, including toxins neutralization, virulence factors inhibition, complement-mediated killing activity, and opsonic phagocytosis. Toxins and bacterial surface components are good targets to generate antibodies against. The U.S. FDA has approved three monoclonal antibody drugs, and there are numerous candidates in the preclinical or clinical trial stages. This article reviews recent advances in the research and development of anti-bacterial monoclonal antibody drugs in order to provide a valuable reference for future studies in this area. KEY POINTS: • Novel drugs against antibiotic-resistant superbugs are urgently required • Monoclonal antibodies can treat bacterial infections through multiple mechanisms • There are many anti-bacterial monoclonal antibodies developed in recent years and some candidates have entered the preclinical or clinical stages of development.

A homodimeric IL-15 superagonist F4RLI with easy preparation, improved half-life, and potent antitumor activities.

Interleukin-15 (IL-15) is a promising candidate for cancer immunotherapy due to its potent immune-activating effects. There are several IL-15 molecules currently in clinical trials but facing shortages of poor half-life, circulation instability, or complicated production and quality control processes. The aim of this study is to design a novel IL-15 superagonist to set out the above difficulties, and we constructed F4RLI consisting of the GS-linker spaced IgG4 Fc fragment, soluble IL-15 Rα (sIL-15Rα), and IL-15(N72D). Using a single plasmid transient transfection in HEK293E cells, the matured F4RLI was secreted in the form of homodimer and got purified by an easy step of protein A affinity chromatography. The F4RLI product can significantly stimulate the proliferation of human CD3+CD8+ T cells and NK cells in vitro. Meanwhile, F4RLI greatly extended the half-life and prolonged the exposure of IL-15 in mice nearly by 28- and 200-fold, respectively, in comparison with that of the IL-15 monomer. In vivo, F4RLI vastly expanded mouse splenic CD8+ T lymphocytes, illustrating its potential in tumor immunotherapy. Further studies showed that the combination of F4RLI with the immune checkpoint blocker atezolizumab played a synergistic effect in treating MC38 mouse tumor by increasing the percentage of CD8+ T cells in tumor tissue. Moreover, the combination therapy of F4RLI with the angiogenesis inhibitor bevacizumab resulted in significant tumor growth suppression in a xenograft human HT-29 mouse model. Overall, our results demonstrate a homodimeric IL-15 superagonist F4RLI with advances in manufacturing processes and biopharmaceutical applications for cancer immunotherapy. KEY POINTS: • The homodimeric structure of F4RLI facilitates its easy production processes and quality control. • The fusion with Fc and sIL-15Rα extends the plasma half-life of IL-15 by about 28-fold. • F4RLI can play synergistic antitumor activity with the PD-1/PD-L1 checkpoint inhibitor or angiogenesis inhibitor.

Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies.

Rapid and efficient bispecific antibody (BsAb) production for industrial applications is still facing many challenges. We reported a technology platform for generating bispecific IgG antibodies, "Bispecific Antibody by Protein Trans-splicing (BAPTS)." While the "BAPTS" method has shown potential in high-throughput screening of BsAbs, further understanding and optimizing the methodology is desirable. A large number of BsAbs were selected to illustrate the conversion efficiency and kinetics parameters. The temperature of reaction makes no significant influence in conversion efficiency, which can reach more than 70% within 2 h, and CD3 × HER2 BsAb can reach 90%. By fitting trans-splicing reaction to single-component exponential decay curves, the apparent first-order rate constants at a series of temperatures were determined. The rate constant ranges from 0.02 to 0.11 min-1 at 37 °C, which is a high rate reported for the protein trans-splicing reaction (PTS). The reaction process is activated rapidly with activation energy of 8.9 kcal·mol-1 (CD3 × HER2) and 5.2 kcal·mol-1 (CD3 × EGFR). The BsAbs generated by "BAPTS" technology not only had the similar post-translation modifications to the parental antibodies, but also demonstrated excellent in vitro and in vivo bioactivity. The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach. KEY POINTS: • The trans-splicing reaction of Npu DnaE intein in "BAPTS" platform is a rapid process with low reaction activation and high rate. • The BsAb generated by "BAPTS" remained effective in tumor cell killing. • The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach.

Strain effect on adsorption and reactions of AHx (A = C, N, O, x ≤ 3) on In2O3(110), TiO2(110), and ZrO2(101) surfaces.

More and more attention has been paid to strain-based regulation of catalytic activity. To guide regulation of catalytic performance via strain engineering, adsorption and reactions of AHx (A = C, N, O, x ≤ 3) were investigated on uniformly strained In2O3 (110), rutile TiO2 (110), and tetragonal ZrO2 (101) from -2% to 4%. The results show that adsorption energies vary linearly with strain; expansive strain enhances the adsorption of most adsorbates. Unlike the adsorbate scaling relations that are central atom dependent, the adsorbate scaling relations on strained surfaces are central atom independent. C-H/O-H bonds are elongated/shortened with expansive strain, and adsorption energies of CHx generally change more than those of OHx and NHx, which can be rationalized with effective medium theory and pertinent bond energies. Thermodynamically, In2O3(110)/ZrO2(101) is the most active/inactive. The estimated variation of rate constants at 300 K from 0% to 2% strain based on the Brønsted-Evans-Polanyi relationship demonstrates great strain regulation potential of catalytic performance on these oxide surfaces. Finally, it is demonstrated that strain tends to facilitate the reactions whose sum of the stoichiometric number is positive, which can be used as a rule to guide strain engineering for heterogeneous catalysis.

Stabilizing the Exotic Carbonic Acid by Bisulfate Ion.

Carbonic acid is an important species in a variety of fields and has long been regarded to be non-existing in isolated state, as it is thermodynamically favorable to decompose into water and carbon dioxide. In this work, we systematically studied a novel ionic complex [H2CO3·HSO4]- using density functional theory calculations, molecular dynamics simulations, and topological analysis to investigate if the exotic H2CO3 molecule could be stabilized by bisulfate ion, which is a ubiquitous ion in various environments. We found that bisulfate ion could efficiently stabilize all the three conformers of H2CO3 and reduce the energy differences of isomers with H2CO3 in three different conformations compared to the isolated H2CO3 molecule. Calculated isomerization pathways and ab initio molecular dynamics simulations suggest that all the optimized isomers of the complex have good thermal stability and could exist at finite temperatures. We also explored the hydrogen bonding properties in this interesting complex and simulated their harmonic infrared spectra to aid future infrared spectroscopic experiments. This work could be potentially important to understand the fate of carbonic acid in certain complex environments, such as in environments where both sulfuric acid (or rather bisulfate ion) and carbonic acid (or rather carbonic dioxide and water) exist.

Protective effects of anti-alginate monoclonal antibody against Pseudomonas aeruginosa infection of HeLa cells.

Pseudomonas aeruginosa is an opportunistic pathogen that is highly resistant to antibiotics, especially when it grows in biofilms. As an alternative to antibiotic intervention, antimicrobial antibody drugs have drawn attention in recent years due to their immunotherapeutic functions. In this study, we designed a monoclonal scFv-Fc-form antibody, MFb, targeting P. aeruginosa antigen alginate and investigated its function against this bacterium in vitro. MFb was generated by transient gene expression in HEK293 cells and purified by one-step protein A affinity chromatography. Experiments showed that MFb could recognize alginate specifically based on enzyme-linked immunosorbent assays. Its KD value of 8.31 nM was determined by surface plasmon resonance, demonstrating its high affinity for alginate. Further detailed studies revealed that the antibody exerted antibacterial effects by three mechanisms: 1) MFb inhibited P. aeruginosa biofilm formation with an IC50 of 0.58 µg/mL; 2) MFb reduced P. aeruginosa adhesion to HeLa cells, and successfully prevented its invasion on epithelial cells; 3) based on an in vitro macrophage phagocytosis assay, MFb could enhance the phagocytotic capacity of macrophages for P. aeruginosa in a concentration-dependent manner. Taken together, our work demonstrated that the antimicrobial monoclonal antibody MFb has a protective effect on HeLa cells, and it may be a promising novel strategy to treat P. aeruginosa infection.

Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies.

High product purity, preserving natural IgG architecture, and excellent production efficiency are highly desirable in bispecific antibody manufacturing. We have reported a platform called Bispecific Antibody by Protein Trans-Splicing (BAPTS) to synthesize BsAbs with natural human IgG structure and no chain mispairing. In the method, two antibody fragments carrying different target-specificities are separately expressed in mammalian cells and subsequently fused to form BsAbs by utilizing the trans-splicing property of the split intein Npu DnaE. The hinge region of antibody, a region with less functional impact, is selected for conjugating the two fragments. The method involves the following steps: (i) constructing five plasmids coding antibody components; (ii) separately expressing and purifying two antibody fragments A and B. Fragment A contains one Fab, "Knobs-into-Holes" mutations in the CH3 domain and NPU DnaEC. Fragment B contains another Fab and NPU DnaEN; (iii) mixing of fragments A and B under permissive reducing conditions in vitro to enable trans-splicing reaction; (iv) removing the reductant to allow re-oxidation of disulfide bonds; (v) isolating BsAb product from unreacted precursors by affinity chromatography. The method allows correct assembly of two heavy and two light chains to form bispecific IgG antibodies in natural structure with no synthetic linkers. No chain mispairing was observed in the product by UPLC-MASS. In addition, the observed kinetics and low reaction activation energy confirmed that the trans-splicing is thermodynamically favored reaction. The BAPTS technology is feasible for industrial applications.

Antibody glycosylation: impact on antibody drug characteristics and quality control.

Glycosylation is a common post-translational modification that occurs during the production of antibodies. Glycans attached to antibodies play an important role in the pharmacokinetics, efficacy, and safety of therapeutic antibodies. In the modern antibody industry, it is important to adjust and control glycosylation modifications. The formation of specific sugar structures via glycosylation engineering is constantly evolving. This review summarizes the recent progress in glycosylation modifications, as well as the major discoveries and current understanding of the mechanisms involved, to provide new ideas for the research and development of therapeutic antibodies.

Concentrations and distribution of novel brominated flame retardants in the atmosphere and soil of Ny-Ålesund and London Island, Svalbard, Arctic.

Novel brominated flame retardants (NBFRs) were investigated in Arctic air and soil samples collected from Ny-Ålesund and London Island, Svalbard, during Chinese scientific research expeditions to the Arctic during 2014-2015. The concentrations of Σ9NBFRs in the Arctic air and soil were 4.9-8.7 pg/m3 (average 6.8 pg/m3) and 101-201 pg/g dw (average 150 pg/g dw), respectively. The atmospheric concentration of hexabromobenzene (HBB) was significantly correlated with that of pentabromotoluene (PBT) and pentabromobenzene (PBBz), suggesting similar source and environmental fate in the Arctic air. No significant spatial difference was observed among the different sampling sites, both for air and soil samples, indicating that the effects of the scientific research stations on the occurrence of NBFRs in the Arctic were minor. The fugacities from soil to air of pentabromoethylbenzene (PBEB), 2,3-dibromopropyl 2,4,6-tribromophenyl ether (DPTE), and decabromodiphenylethane 1,2-bis (pentabromophenyl) ethane (DBDPE) were lower than the equilibrium value, indicating a nonequilibrium state of these compounds between air and soil, the dominant impact of deposition and the net transport from air to soil. The correlation analysis between the measured and predicted soil-atmosphere coefficients based on the absorption model showed that the impact of the soil organic matter on the distribution of NBFRs in the Arctic region was minor. To the best of our knowledge, this work is one of the limited reports on atmospheric NBFRs in the Arctic and the first study to investigate the occurrence and fate of NBFRs in the Arctic soil.

Molecular dynamics based improvement of the solubilizing self-cleavable tag Zbasic-ΔI-CM application in the preparation of recombinant proteins in Escherichia coli.

Zbasic-ΔI-CM is a novel intein-based self-cleavable tag we developed to accelerate the soluble expression of recombinant proteins in Escherichia coli (E. coli). Previously we found that intein activity could be interfered by its flanking exteins, and thus reducing the production efficiency and final yield. In this work, we used CXC-chemokine 9 (CXCL9) as a model C-extein, which fusion with Zbasic-ΔI-CM showed high intein activity. When the fusion protein got soluble expression, CXCL9 was released immediately and purified directly from cell lysis supernatant. The results demonstrated that Zbasic-ΔI-CM tag had successfully mediated the efficient production of high-quality CXCL9 with reduced time and resources consumption in comparison with inclusion bodies expression. Molecular dynamics simulations suggested that the improved cleavage activity of Zbasic-ΔI-CM upon fusion with CXCL9 may be due to the higher dynamics of the first half loop and stabilization of the second half loop of intein. Our results proved that the self-cleavable Zbasic-ΔI-CM mediated soluble expression could be a feasible process for cytokines like CXCL9, thus of attractive potentials for production of therapeutic proteins using E. coli expression system.

A general platform for efficient extracellular expression and purification of Fab from Escherichia coli.

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.

Feasible development of stable HEK293 clones by CRISPR/Cas9-mediated site-specific integration for biopharmaceuticals production.

OBJECTIVE: To inspect the feasibility of recombinant stable HEK293 cell lines development for biopharmaceuticals production using CRISPR/Cas9-mediated site-specific integration. RESULTS: Using EGFP as a model protein, we first confirmed that the 'safe harbor' AAVS1 locus could be successfully targeted and the exogenous genes could be integrated through homology-directed repair induced by CRISPR/Cas9 technology. Then we constructed a donor plasmid harboring CTLA4Ig gene with an upstream CMV promoter and a downstream puromycin N-acetyltransferase gene to accelerate the efficient integration and selection of CTLA4Ig expression clones. After puromycin enrichment, the transfected pool was diluted for single clone selection, and 12 recombinant clones with CTLA4Ig expression were finally selected with a targeting efficiency of 25.8%. Productivity assay demonstrated that a frequency of 83.3% of selected clone were of consistent productivities, thus illustrating the high efficiency and success rate of this strategy. CONCLUSIONS: CRISPR/Cas9 mediated site-specific integration is an efficient and reliable tool to establishment recombinant stable HEK293 cell lines for both academic and industrial applications.

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

A novel self-cleavable tag Zbasic-∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli.

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic-intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein "Zbasic-∆I-CM-IL-15" was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic-∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC50 was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic-∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.