Expression of recombinant apopholasin using a baculovirus–silkworm multigene expression system and activation via dehydrocoelenterazine

Pholasin is a photoprotein derived from the glowing bivalve mollusk, Pholas dactylus. Even though the chemical structure of the prosthetic group (chromophore) responsible for the light emission character of the mollusk remains unknown, research has shown that the presence of dehydrocoelenterazine (DCL) increased light emission and that the dithiothreitol adduct of DCL was isolated from Pholasin®. To date, our research has been focused on activating apopholasin, the naturally occurring apoprotein of Pholasin®, using DCL. In the current study, the expression of recombinant apopholasin via a baculovirus–silkworm multigene expression system is reported. Additionally, the purification of apopholasin using a Flag®-affinity column, the activation of apopholasin using DCL, and the initiation of its luminescent character through the addition of a peroxidase–hydrogen peroxide mixture are reported. The peroxidase–H₂O₂-dependent luminescence was observed from the recombinant apopholasin activated with DCL.     

Production of membrane proteins using cell–free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering C_H3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.     

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering C_H3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.     

Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system

The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult‐to‐express proteins. For this reason, we evaluated the more recently established novel CAP‐T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP‐T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. Biotechnol. Bioeng. 2012;109: 2250–2261. © 2012 Wiley Periodicals, Inc.     

Optimization of the production of triabin, a novel thrombin inhibitor, in High Five™ insect cells infected with a recombinant baculovirus

The isolation of a new type of thrombin inhibitor, called triabin, from the saliva of the hematophagous bug Triatoma pallidipennis, has recently been described. In the in vitro platelet aggregation inhibition assay triabin has a similar potency as the thrombin inhibitor hirudin now in phase III clinical trials. However, in another in vitro assay using a low molecular weight substrate for thrombin, triabin does not inhibit thrombin completely even at 6 fold higher molar doses in comparison with hirudin. This means that triabin has a novel mode of action towards thrombin making triabin into an interesting candidate as a therapeutic agent. Recently it has been shown that a recombinant baculovirus can be efficiently used for the triabin production in insect cells and that the yields in adherent cultures of High Five™ cells (approx. 20 mg l-1) were about 7 fold higher than in adherent cultures of Sf9 cells (approx. 3 mg l- 1). To optimize the triabin yield from the baculovirus/insect cell expression system, experiments were performed with suspension adapted cultures of High Five™ cells to investigate the effects of the state of the host cell, of the multiplicity of infection, of the cell density at the time of infection and of supplementation of the medium with nutrients and oxygen. Triabin yields of up to 200 mg l-1, as determined by an activity assay, could finally be obtained here. This revised version was published online in July 2006 with corrections to the Cover Date.     

In search of expression bottlenecks in recombinant CHO cell lines—a case study

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells’ quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell’s productivity.     

Production characteristics of interferon-α using an l-arabinose promoter system in a high-cell-density culture

 Using high-cell-density culture of Escherichia coli under the control of an l-arabinose promoter (P_araB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied. The inducer, l-arabinose, showed a maximal induction level above 10.7 mM in the final concentration. The concentration of inducer also affected the partition of interferon-α (IFN-α) into the soluble form and inclusion bodies. Induction kinetics of the rate of accumulation of IFN-α on the P_araB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac. These innate characteristics of P_araB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein. The duration time of induction could control the expression of both soluble and insoluble protein. The ratio of yeast extract to glycerol (N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation. The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion bodies in the total expressed IFN-α. Received: 21 May 1999 / Received last revision: 16 August 1999 / Accepted: 2 September 1999     

Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin–SUMO dual fusion system

LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in host defense. In this report, the peptide was expressed as a fusion with a thioredoxin–SUMO dual-tag. Upon SUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus was generated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusion chromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecular weight as theoretically expected for its native form. The produced peptide displayed antimicrobial activity against Escherichia coli K-12. On average, 2.4 mg peptide was obtained from one liter of bacterial culture. Thus, the described approach provides an effective alternative for producing active recombinant LL-37 with its natural amino acid sequence in E. coli.     

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with -fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO₃. Glucose, an inhibitor of -fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and -fructofuranosidase from Asp. niger.     

Low expression of TMPRSS2–a SARS-CoV-2 internalization protease–associates with basal subtype of head and neck squamous cell carcinoma

SARS-CoV-2 is a single-stranded RNA virus that has caused the ongoing COVID-19 pandemic. ACE2 and other genes utilized by SARS-CoV-2 to enter human cells have been shown to express in Head and Neck Squamous Cell Carcinoma (HNSCC) patients. However, their expression pattern in different subtypes has not been investigated. Hence, in the current study, we have analyzed the expression of ACE2, TMPRSS2 and FURIN in 649 HNSCC patients from two independent cohorts. Our analysis showed significantly lower expression of TMPRSS2 while significantly increased expression of ACE2 and FURIN in HPV-negative HNSCC. Comparison of expression of these genes in the three subtypes of HNSCC patients (basal, classical and inflamed/mesenchymal) showed no significant difference in the expression of ACE2 among the three subtypes; however, the basal subtype showed significantly reduced expression of TMPRSS2 but significantly increased expression of FURIN. Comparison of expression of these genes between the HPV-negative patients of basal subtype vs all others confirmed significantly lower expression of TMPRSS2 in HPV-negative patients of basal subtype as compared to all others. Our study shows that the different subtypes of HNSCC patients have different expression patterns of genes utilized by the SARS-CoV-2 to enter human cells, and hence, their susceptibility to SARS-CoV-2 may also be different. As the expression of TMPRSS2 is significantly lower in the HNSCC patients of the basal subtype, we predict that these patients would be less susceptible to SARS-CoV-2 infection than the patients of other subtypes. However, these findings need to be further validated.     

Evaluation of hydrogen bonds of ecdysteroids in the ligand–receptor interactions using a protein modeling system

The insect molting hormone, 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) specifically bind to the ecdysone receptor. Previously, we synthesized various ecdysteroids containing the side chain moiety of ponasterone A (PonA), and measured the binding activity against Drosophila Kc cells to study the structure–activity relationship. Here we quantitatively analyzed the structure–activity relationship for the ligand binding of ecdysteroids including 20E and PonA. Since the hydrogen bonding (HB) is one of the important physicochemical properties for ligand binding to the ecdysteroid receptor, the number of possible HBs between the ligand molecule and the receptor was manually counted in the modeled ligand–receptor complex for all compounds. The construction of the ligand–receptor model was executed by the full-automatic modeling system (FAMS) in which calculation was done by simulated annealing. The binding potency of 15 ecdysteroids to Kc-cells were linearly correlated (r² = 0.63) with the number of HBs which are observed between ligand and receptor molecule. Contribution of steric and electrostatic effects on the ligand–receptor binding was also examined using a three-dimensional quantitative structure–activity relationship (3-D QSAR), comparative molecular field analysis (CoMFA).     

High-level expression of a recombinant fragment of human fibronectin containing the Cell I–Hep II–IIICS71 domain in Escherichia coli as a soluble protein

Fibronectin (FN) is a major matrix protein that is involved in multiple processes. Its Cell I–Hep II domain is potentially useful in tumor therapy. Here, a recombinant fragment of FN with the Cell I–Hep II-IIICS71 domain, CH/71, was expressed in Escherichia coli. The CH/71 fusion protein consists of Cell I–Hep II domain and 19th to 89th amino acids of IIICS domain of FN. The expression level of CH/71 in E. coli was very high after induction with IPTG. Furthermore, CH/71 protein was largely found in the soluble fraction. It was readily purified by one-step heparin–agarose affinity chromatograph. The ability of CH/71 binding cells was about 8-fold of that of Cell I–Hep II domain FN.     

Quantification of diadenosine polyphosphates in blood plasma using a tandem boronate affinity–ion exchange chromatography system

Endogenous diadenosine polyphosphates (Ap_nAs) have been associated with a variety of biological effects but quantifying their concentration in blood is difficult. We report on the development of a tandem affinity–ion exchange high-performance liquid chromatography (HPLC) system that employs boronate affinity upstream of ion exchange chromatography for automated rapid (45-min) resolution and extraction of Ap_nAs from human plasma. This system obviates previous requirements for multiple column separations and handling steps, so it is ideally set up for time- and cost-efficient screening of blood samples for Ap_nA pharmacokinetic and biodistribution studies.     

High level expression of a frog α-amidating enzyme,AE-II,in cultured cells and silkworm larvae using aBombyx mori nuclear polyhedrosis virus expression vector

AXenopus laevis peptidyl C-terminal α-amidating enzyme (AE-II) gene, modified by deletion of a region encoding the putative membrane-spanning domain and the putative C-terminal cytosolic tail, was expressed in BoMo-15 AIIc insect cells and silkworm larvae using aBombyx mori baculovirus expression vector system. The expressed enzyme was identified predominantly in the culture medium and the hemolymph of silkworm larvae, indicating successful secretion of the expressed AE-II. The level of recombinant enzyme in the larval hemolymph at 4 days post-infection (40 μg/ml) was more than 100-fold the peak levels found in the culture medium (250 ng/ml). The enzyme activity in the larval hemolymph at 4 days post-infection was 3700 units/ml.     

Influence of aeration–homogenization system in stirred tank bioreactors,dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO₂ and NaHCO₃ solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO₃ solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10⁶ cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation–aeration systems.     

Baculovirus immediate-early promoter-mediated expression of the Zeocin™ resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 μg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 μg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3–4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by β-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.     

Insights into unbound–bound states of GPR142 receptor in a membrane-aqueous system using molecular dynamics simulations

G protein coupled receptors (GPCRs) are source machinery in signal transduction pathways and being one of the major therapeutic targets play a significant in drug discovery. GPR142, an orphan GPCR, has been implicated in the regulation of insulin, thereby having a crucial role in Type II diabetes management. Deciphering of the structures of orphan, GPCRs (O-GPCRs) offer better prospects for advancements in research in ion translocation and transduction of extracellular signals. As the crystallographic structure of GPR142 is not available in PDB, therefore, threading and ab initio-based approaches were used for 3D modeling of GPR142. Molecular dynamic simulations (900 ns) were performed on the 3D model of GPR142 and complexes of GPR142 with top five hits, obtained through virtual screening, embedded in lipid bilayer with aqueous system using OPLS force field. Compound 1, 3, and 4 may act as scaffolds for designing potential lead agonists for GPR142. The finding of GPR142 MD simulation study provides more comprehensive representation of the functional properties. The concern for Type II diabetes is increasing worldwide and successful treatment of this disease demands novel drugs with better efficacy.     

Fast screening method for benzophenanthridine alkaloids in plant cell cultures of California poppy using a dansyl-glycine-β-cyclodextrin molecular recognition system

The association constant K for the host-guest complex comprised of sanguinarine/dansyl-glycine--cyclodextrin was found to be 5.75 × 105 M. The sensitivity factor(), which is defined as the ratio of the change of the fluorescence intensity after complex formation to the intensity of free, uncomplexed dansyl-glycine--cyclodextrin, showed an almost linear dependence on analyte concentration up to 50M sanguinarine. This molecular recognition system can be exploited to rapidly estimate the total concentration of benzophenanthridine alkaloids in suspension cultures of Eschscholtzia californica. © Rapid Science Ltd. 1998     

Preparation of nitrophorin 7(Δ1–3) from Rhodnius prolixus without start–methionine using recombinant expression in Escherichia coli

The heterologous recombinant expression of proteins in Escherichia coli without start–methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB–nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl₂ gradient.