Selenoproteins via genetic code expansion in mammalian cells

Many open questions remain in the field of selenoprotein biology, including the functions of a number of uncharacterized human selenoproteins, and the properties of selenocysteine compared to its analogous amino acid cysteine. The mechanism of selenocysteine incorporation involves an intricate machinery that deviates from the mechanism of incorporation for the canonical 20 amino acids.
As a result, recombinant expression of selenoproteins has been historically challenging, and has hindered a deeper evaluation of selenoprotein biology. Genetic code expansion methods, which incorporate protected analogs of selenocysteine, allow the endogenous selenocysteine incorporation mechanism to be bypassed entirely to facilitate selenoprotein expression.
Here we present a method for incorporating a photocaged selenocysteine amino acid (DMNB-Sec) into human selenoproteins directly in mammalian cells. This approach offers the opportunity to study human selenoproteins in their native cellular environment and should advance our understanding of selenoprotein biology.

Nitroreduction of flutamide by Cunninghamella elegans NADPH: Cytochrome P450 reductase

The microbial model of mammalian drug metabolism, Cunninghamella elegans, has three cytochrome P450 reductase genes in its genome: g1631 (CPR_A), g4301 (CPR_B), and g7609 (CPR_C). The nitroreductase activity of the encoded enzymes was investigated via expression of the genes in the yeast Pichia pastoris X33. Whole cell assays with the recombinant yeast demonstrated that the reductases converted the anticancer drug flutamide to the nitroreduced metabolite that was also produced from the same substrate when incubated with human NADPH: cytochrome P450 reductase.
The nitroreductase activity extended to other substrates such as the related drug nilutamide and the environmental contaminants 1-nitronaphthalene and 1,3-dinitronaphthalene. Comparative experiments with cell lysates of recombinant yeast were conducted under aerobic and reduced oxygen conditions and demonstrated that the reductases are oxygen sensitive.

Recombinant Protein Production and Purification of Insoluble Proteins

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The efficient production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation.
Thus, the selection of the optimal expression organism and its growth conditions to minimize the formation of insoluble protein aggregates should be done according to the protein characteristics and downstream requirements.
Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293T cells in both culture formats. DMEM was the best transfection medium for adherent HEK 293T cells, so we determined the kinetics of linear polyethyleneimine (LPEI) polyplex formation with plasmid DNA (pDNA) and subsequent cellular uptake. Statistical experimental designs revealed optimal transfection efficiency using 0.7pg pDNA and 4.5pg LPEI per cell. We used the amount of pDNA and LPEI per cell as the transfer criterion for HEK 293T/17SF cell suspension cultures in FreeStyle 293 medium and confirmed optimal transfection at 1.1pg pDNA and 6.6pg LPEI per cell.
We observed a strong correlation between polyplex size, transfection efficiency and post-transfection cell viability. Suspension cell transfection could be scaled to a 100-mL working volume without loss of efficiency. We conclude that pg pDNA and pg LPEI per cell is a suitable transfer criterion allowing the optimization of transient transfection using statistical experimental designs, thus minimizing the amount of pDNA and LPEI used without sacrificing transfection efficiency.

In vitro pharmacological profile of PHA-022121, a small molecule bradykinin B 2 receptor antagonist in clinical development

PHA-022121 is a novel small molecule bradykinin B2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B2 receptor with Ki values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B2 receptor with Kb values of 0.15 and 0.26 nM, respectively (calcium mobilization assay).
Antagonist potency at the recombinant cynomolgus monkey bradykinin B2 receptor is similarly high (Kb values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B2 antagonist activity with pA2 values of 0.35 and 0.47 nM, respectively.
The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B2 receptor as assessed using recombinant or endogenously expressed receptors.

Nanoscale Mapping of Recombinant Viral Proteins: From Cells to Virus-Like Particles

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFlu-S). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed.
Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

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