Mammalian Expression System Research Articles

Structural, functional, and evolutionary differences between PD-L1 and PD-L2

Abstract The PD-1/PD-L1/PD-L2 axis is a critical immune checkpoint that tips immune responses towards tolerance. This axis has been heavily targeted in cancer immunotherapy with monoclonal antibodies, which block receptor-ligand interactions. PD-L1 is constitutively expressed at low levels and is induced on nearly all tissues upon IFN-gamma signaling. In contrast, PD-L2 expression is restricted mainly to antigen presenting cells (APC)s, such as dendritic cells and macrophages. PD-L2 has a 4-fold stronger affinity for PD-1 than does PD-L1. It has been proposed that W110 of PD-L2 accounts for this affinity difference; the corresponding PD-L1 residue is A121. To test this hypothesis, we produced, in a mammalian expression system, a set of PD-L1 and PD-L2 proteins with the W and A swapped, and studied their binding to PD-1 using surface plasmon resonance. Surprisingly, rather than decreasing affinity as expected, a W110A substitution improved affinity by decreasing the off-rate. We propose a novel structural mechanism for PD-L2’s affinity advantage, one that interestingly emerged upon the evolution of placental mammals. We examined the functional effect that these structural alterations have on the PD-Ligands ability to inhibit T cell activation and found a correlation. Additionally, we discovered that PD-Ligands homo- and hetero-dimerize in a manner similar to other B7-family members, with PD-L2 displaying the strongest homo-dimerization. Collectively, these data support our hypothesis that PD-L2 expression on APCs provides an inhibitory advantage, one that may heighten the tolerogenic hurdle that an immune response must overcome during the priming phase.

Development of a Tuberculosis Vaccine Seed: Construction of Resuscitation-Promoting Factor B DNA Vaccine and its Expression in Vitro and in Vivo

Background: Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (Mtb) and has a high death-rate worldwide. Bacillus Calmette-Guerin is the only TB vaccine which is currently available with several drawbacks, such as its different efficacy for different individuals, lack of protection for lung TB in adults and subsequent reactivation which lead the research for novel TB vaccine approach. Resuscitation-promoting factor (rpf) protein in Mtb is a protein cluster which play a big role in TB dormancy during latent infection. Member from this cluster protein is rpfB which shows the greatest biological and immunological characteristics among other proteins in the rpf family, now is widely explored as novel TB vaccine candidate. Methods: In this study, the rpfB gene of the Mtb Beijing strain was amplified using PCR and then cloned into pcDNA3.1 plasmids. The ability of recombinant pcDNA-rpfB to induce humoral immune response was tested through Balb/C mice immunization. Results: A positive recombinant rpfB protein ~66 kDa was detected through western blot analysis using immunized mice sera. Meanwhile, recombinant pcDNA-rpfB was transfected in to CHO-K1 mammalian cell line and recombinant rpfB antigen expression was confirmed through immunostaining. Conclusions: Therefore, we have succesfully express the recombinant rpfB proten of M.tb strain Beijing in mammalian expression system which proven to be antigenically induced humoral immune response in mice model.

Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV)

Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism.

ABSTRACTHaploinsufficiency of DYRK1A is a cause of a neurodevelopmental syndrome termed mental retardation autosomal dominant 7 (MRD7). Several truncation mutations, microdeletions and missense variants have been identified and result in a recognizable phenotypic profile, including microcephaly, intellectual disability, epileptic seizures, autism spectrum disorder and language delay. DYRK1A is an evolutionary conserved protein kinase which achieves full catalytic activity through tyrosine autophosphorylation. We used a heterologous mammalian expression system to explore the functional characteristics of pathogenic missense variants that affect the catalytic domain of DYRK1A. Four of the substitutions eliminated tyrosine autophosphorylation (L245R, F308V, S311F, S346P), indicating that these variants lacked kinase activity. Tyrosine phosphorylation of DYRK1A-L295F in mammalian cells was comparable to wild type, although the mutant showed lower catalytic activity and reduced thermodynamic stability in cellular thermal shift assays. In addition, we observed that one variant (DYRK1A-T588N) with a mutation outside the catalytic domain did not differ from wild-type DYRK1A in tyrosine autophosphorylation, catalytic activity or subcellular localization. These results suggest that the pathogenic missense variants in the catalytic domain of DYRK1A impair enzymatic function by affecting catalytic residues or by compromising the structural integrity of the kinase domain.This article has an associated First Person interview with the first author of the paper.

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purifiedhEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

Visualisation of intracellular production bottlenecks in suspension-adapted CHO cells producing complex biopharmaceuticals using fluorescence microscopy

With the advance of complex biological formats such as bispecific antibodies or fusion proteins, mammalian expression systems often show low performance. Described determining factors may be accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. In case of the investigated bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cell line neither impaired transcription nor decreased translation processes were identified and thus satisfactorily explained its low production capacity. Hence, we established a streamlined confocal microscopy-based methodology for CHO production cells investigating the distribution of the recombinant protein within the respective organelles of the secretory pathway and visualised the structure of the endoplasmic reticulum (ER) to be affected pinpointing towards an intra-ER bottleneck putatively hampering or limiting efficient secretion. The ER displayed not only a heavily altered morphology in comparison to a high immunoglobulin G (IgG)-producing cell line with a possibly inflated or overloaded structure, but the recombinant protein was also completely absent in the Golgi apparatus. Notably, the results obtained using an automated microscopy approach suggest the possible application of this methodology in cell line development and engineering.

Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.

Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinct N-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrate-protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed.

G-Protein Beta-Gamma Subunits Inhibit the Heat-Sensitive TRPM3 Ion Channels

Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated non-selective, Ca2+ permeable cation channel also stimulated by chemical agents such as pregnenolone sulphate and CIM0216. Here we show that activation of Gi-coupled cell surface receptors inhibits TRPM3 currents in a mammalian expression system, which was alleviated by co-expression of proteins that bind βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβ1γ2, Gβ3γ2, Gβ4γ2, but not Gβ5γ2 or constitutively active mutants of Gαo or Gαi, inhibited pregnenolone sulphate-induced TRPM3 currents. Purified Gβγ proteins applied to excised inside out patches also inhibited TRPM3 activity, indicating a direct effect. Baclofen, somatostatin, and DAMGO, agonists of Gi coupled receptors, inhibited Ca2+ signals induced by pregnenolone sulphate and CIM0216 in dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited CIM0216-induced currents in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in vivo. Our data show that Gβγ inhibits TRPM3 channels upon Gi-coupled receptor activation.

Functional characterization of oligopeptide transporter 1 of dairy cows

BackgroundIt is well known that peptides play a vital role in the nutrition and health of dairy cows. Bovine oligopeptide transporter 1 (bPepT1) is involved in the peptide transport process in the gastrointestinal tracts of dairy cows. However, little information is known in the characteristics of bPepT1. Therefore, the purpose of this study was to characterize bPepT1 functionally using a mammalian cell expression system. The uptake of radiolabeled dipeptide glycyl-sarcosine ([3H]-Gly-Sar) into the bPepT1-transfected Chinese hamster ovary cells was measured at various pH and substrate concentrations and with or without 15 other small peptides that contained Met or Lys.ResultsWestern blot results showed that the abundance of bPepT1 protein in the jejunum and ileum are the highest in the gastrointestinal tract of dairy cows. The uptake of [3H]-Gly-Sar by bPepT1-Chinese hamster ovary cells was dependent on time, pH, and substrate concentration, with a low Km value of 0.94 ± 0.06 mmol/L and a maximum velocity of 20.80 ± 1.74 nmol/(mg protein • 5 min). Most of the di- and tripeptides were the substrates of bPepT1, based on substrate-competitive studies. However, bPepT1 has a higher affinity to the peptides with shorter chains, greater hydrophobicity, and negative or neutral charges.ConclusionsThese results demonstrated for the first time the functional characteristics of bPepT1, and they provide a new insight and better understanding into its vital role in absorbing a wide range of peptides from the digestive tract of dairy cows.

Glycoengineering of Mammalian Expression Systems on a Cellular Level.

Mammalian expression systems such as Chinese hamster ovary (CHO), mouse myeloma (NS0), and human embryonic kidney (HEK) cells serve a critical role in the biotechnology industry as the production host of choice for recombinant protein therapeutics. Most of the recombinant biologics are glycoproteins that contain complex oligosaccharide or glycan attachments representing a principal component of product quality. Both N-glycans and O-glycans are present in these mammalian cells, but the engineering of N-linked glycosylation is of critical interest in industry and many efforts have been directed to improve this pathway. This is because altering the N-glycan composition can change the product quality of recombinant biotherapeutics in mammalian hosts. In addition, sialylation and fucosylation represent components of the glycosylation pathway that affect circulatory half-life and antibody-dependent cellular cytotoxicity, respectively. In this chapter, we first offer an overview of the glycosylation, sialylation, and fucosylation networks in mammalian cells, specifically CHO cells, which are extensively used in antibody production. Next, genetic engineering technologies used in CHO cells to modulate glycosylation pathways are described. We provide examples of their use in CHO cell engineering approaches to highlight these technologies further. Specifically, we describe efforts to overexpress glycosyltransferases and sialyltransfereases, and efforts to decrease sialidase cleavage and fucosylation. Finally, this chapter covers new strategies and future directions of CHO cell glycoengineering, such as the application of glycoproteomics, glycomics, and the integration of 'omics' approaches to identify, quantify, and characterize the glycosylated proteins in CHO cells. Graphical Abstract.

Production, Purification, and Characterization of Antibody-TNF Superfamily Ligand Fusion Proteins.

Antibody-fusion proteins with ligands, e.g., of the TNF superfamily (TNFSF) can be adequately produced in mammalian expression systems. Here, we describe the transient production in adherent and suspension human embryonic kidney cells at laboratory scale, followed by purification procedures applying protein A and immobilized metal affinity chromatography for proteins with Fc domain and 6×histidine-tag, respectively. In addition, characterization of the purified proteins by size exclusion chromatography is described.

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line.

Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein. Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE. Result The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%. Conclusions By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets.

Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in conjunction with fluorescent proteins in HeLa-based mammalian CFE system provides a versatile in vitro platform for studying mRNA dynamics for bottom-up synthetic biology applications.

Probing the importance of clonality: Single cell subcloning of clonally derived CHO cell lines yields widely diverse clones differing in growth, productivity, and product quality.

In the past few decades, a large variety of therapeutic antibodies and proteins have been expressed in Chinese hamster ovary (CHO) cells. This mammalian expression system is robust, scalable, relatively inexpensive, and importantly allows for post-translational modifications that are important for some therapeutic proteins. Historically, CHO cell lines were derived from colonies of cells grown in semi-solid or liquid plates using either serum-containing or serum-free media. Current advancements in cell sorting and imaging technologies have allowed for isolating and imaging single cell progenitors at the seeding step, significantly increasing the probability of isolating clonally derived cell lines. However, it is debatable how much population heterogeneity can be eliminated when clonally derived cell lines, originated from a single cell progenitor, are scaled up. To further investigate this phenomenon, we subcloned two different clonally derived (day 0 imaged and visually inspected) cell lines expressing antibody-X. The results showed that when six randomly chosen subclones of each line were evaluated in a production assay, these subclones displayed a range of variation in titer, specific productivity, growth, and product quality attributes. Some subclones displayed variations in transgene copy numbers. Additionally, clonal derivation did not assure stability of the derived cell lines. Our findings show that cell heterogeneity exists in a population even when derived from a single cell progenitor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:624-634, 2018.

Transient expression of human antibodies in mammalian cells.

Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells. Antibody constructs are designed by combining antibody variable domains, generated by phage display or derived from human/humanized monoclonals, with constant regions. The constructs are then expressed from mammalian vectors, secreted into culture media, purified by affinity chromatography and characterized by biolayer interferometry. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin-free antibody reagents suitable for testing in animal models within a 3-week time frame.

Highly motif- and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression

ABSTRACTRecent bioinformatics studies have demonstrated a wide-spread occurrence of the hammerhead ribozyme (HHR) and similar small endonucleolytic RNA motifs in all domains of life. It is becoming increasingly evident that such ribozyme motifs participate in important genetic processes in diverse organisms. Although the HHR motif has been studied for more than three decades, only little is known about the consequences of ribozyme activity on gene expression. In the present study we analysed eight different naturally occurring HHR sequences in diverse genetic and organismal contexts. We investigated the influence of active ribozymes incorporated into mRNAs in mammalian, yeast and bacterial expression systems. The experiments show an unexpectedly high degree of organism-specific variability of ribozyme-mediated effects on gene expression. The presented findings demonstrate that ribozyme cleavage profoundly affect gene expression. However, the extent of this effect varies and depends strongly on the respective genetic context. The fast-cleaving type 3 HHRs [CChMVd(-) and sLTSV(-)] generally tended to cause the strongest effects on intracellular gene expression. The presented results are important in order to address potential functions of naturally occurring ribozymes in RNA processing and post-transcriptional regulation of gene expression. Additionally, our results are of interest for biotechnology and synthetic biology approaches that aim at the utilisation of self-cleaving ribozymes as widely applicable tools for controlling genetic processes.

Recombinant Vaccinia virus-coded interferon inhibitor B18R: Expression, refolding and a use in a mammalian expression system with a RNA-vector.

B18R protein of Vaccinia virus binds to type I interferons and inhibits activation of interferon-mediated signal transduction. Cells which have unimpaired interferon signaling such as primary cell cultures or some industrially important cell lines are capable of development of an antiviral state. An establishment of the antiviral state limits replication of RNA-viruses and can suppress replication of RNA vectors. The interferon inhibitor B18R effectively prevents the establishment of the antiviral state. For this reason, B18R has become a ubiquitous component of protocols for epigenetic reprogramming which use transfections of RNA replicons or mRNA. Despite wide practical applicability, commercially available B18R is predominantly produced in cell cultures and little information has been published on a production and use of bacterially expressed B18R. Objectives of this study were to produce B18R in an E.coli expression system and to confirm the product’s biological activity by using it to maintain RNA-vectors in cell cultures capable of the antiviral state. The described method allows the expression and efficient refolding to obtain 10–100 mg of B18R from a small-scale culture and the production process is economically attractive compared to a use of an eukaryotic expression. To check for a presence of the biological activity of bacterially-expressed B18R the protein was used to support persistence of an autonomously replicating RNA-vector in a cell culture which is capable of the antiviral state. A RNA-containing virus, Venezuelan equine encephalitis virus (VEE) can serve as an efficient vector for heterologous expression in cell cultures, although its replication is sensitive to the effects of type I interferons which limit a range of cell lines for a use with this vector. The VEE replicon was utilized to direct an expression of recombinant human granulocyte colony stimulating factor (G-CSF). The producing replicon could persist in HEK293 cells for sufficiently long time only in presence of B18R, whereas addition of B18R not only allowed persistence of the replicon but also increased production from the replicon. A model product granulocyte colony stimulating factor accumulated to 35.5 μg/ml during a 7 day experiment. This work describes efficacious expression and refolding of the viral cytokine inhibitor and demonstrates a utility of bacterially-expressed B18R.

Human LYPD8 protein inhibits motility of flagellated bacteria

BackgroundWe previously reported that the mouse Ly6/Plaur domain containing 8 (mLypd8), a GPI-anchored protein highly and selectively expressed on colonic epithelia, contributes to segregation of intestinal microbiota and intestinal epithelia and is critical for prevention of intestinal inflammation. In addition, it was found that human LYPD8 (hLYPD8) is expressed in the colonic epithelia and expression of hLYPD8 is reduced in some ulcerative colitis patients. However, the molecular characteristics and functions of hLYPD8 remain unclear. In this study, we generated the hLYPD8 protein and characterized its functions.MethodsTo analyze the characteristics and functions of the hLYPD8 protein, recombinant FLAG-tagged hLYPD8 protein was generated by two kinds of protein expression systems: a mammalian cell expression system and a Pichia pastoris expression system. Recombinant hLYPD8 protein was analyzed by western blot analysis or deglycosylation assay. The effect of the protein on flagellated bacteria was examined by ELISA assay and motility assay using semi-agar plates.ResultshLYPD8 was a highly N-glycosylated GPI-anchored protein, like mLypd8. Moreover, recombinant hLYPD8 protein generated by the Pichia pastoris expression system using the SuperMan5 strain, which enabled production of a large number of proteins with human-like glycosylation, presented the high binding affinity and the motility inhibitory function to flagellated bacteria, such as Proteus mirabilis.ConclusionsThese results demonstrated that hLYPD8 inhibits the motile activity of flagellated bacteria, many of which are involved in intestinal inflammation. The supplementation of recombinant hLYPD8 protein might be a novel therapeutic approach for intestinal inflammation of inflammatory bowel diseases.

Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification

Procollagen type I carboxy-terminal propeptide (PICP), derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin recovery. PICP is a heterotrimeric glycoprotein consisting of two α1 chains (PICPα1) and one α2 chain (PICPα2). Here, we report the recombinant expression of human PICP using a mammalian expression system. Co-expression of PICPα1 and PICPα2 in HEK293F cells resulted in the production of functional PICP in the correctly assembled heterotrimeric form. Using the recombinant PICP as an antigen, we isolated PICP-specific human monoclonal antibodies from phage-displayed antibody libraries and raised rabbit polyclonal antibodies. Using those antibodies, we then developed a sandwich ELISA for PICP with a limit of detection of 1 ng/mL and a measurable range of 1–640 ng/mL. Both intra- and inter-assay imprecision values were <10%. For measuring PICP levels in human fibroblast cellular extracts and culture supernatants and a human serum, the developed ELISA kit displayed comparable performance to that of a commercialized kit. Our results provide an efficient production strategy for recombinant PICP, facilitating the generation of PICP-specific antibodies and development of PICP sandwich ELISA, with potential use in clinical diagnosis of serum samples and testing of cosmeceutical ingredients in fibroblast cell cultures.

TGF-β contamination of purified recombinant GDF15.

Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in purified protein batches may influence the experimental results given that receptors for TGF-β are present. When we attempted to characterize possible receptors for the TGF-β superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-β activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-β and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results.