Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells.

Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.

Nano-Microscopy of Therapeutic Antibody Aggregates in Solution.

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage.

Autoactivation of C-terminally truncated Ca/calmodulin-dependent protein kinase (CaMK) Iδ via CaMK kinase-independent autophosphorylation.

Ca/calmodulin-dependent protein kinase I isoforms (CaMKIα, β, γ, and δ) play important roles in Ca signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1-299) and mCaMKIδ(1-297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E.

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins.

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging.

In-Solution Microscopic Imaging of Fractal Aggregates of a Stressed Therapeutic Antibody.

Aggregates of therapeutic proteins that can contaminate drug products during manufacture is a growing concern for the pharmaceutical industry because the aggregates are potentially immunogenic. Electron microscopy is a typical, indispensable method for imaging nanometer- to micrometer-sized structures. Nevertheless, it is not ideal because it must be performed with ex situ monitoring under high-vacuum conditions, where the samples could be altered by staining and drying.

Suppression of Aggregation of Therapeutic Monoclonal Antibodies during Storage by Removal of Aggregation Precursors Using a Specific Adsorbent of Non-Native IgG Conformers.

The quality of preparations of therapeutic IgG molecules, widely used for the treatment of various diseases, should be maintained during storage and administration. Nevertheless, recent studies demonstrate that IgG aggregation is one of the most critical immunogenicity risk factors that compromises safety and efficacy of therapeutic IgG molecules in the clinical setting. During the IgG manufacturing process, 0.

Characterization of CoPK02, a Ca/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea.

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C.

AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG.

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay.

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials.

High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli.

We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase.

Expression analyses of splice variants of zebrafish cyclin-dependent kinase-like 5 and its substrate, amphiphysin 1.

Mammalian cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase mainly expressed in the central nervous system and believed to be involved in neuronal functions. However, the functions of CDKL5 in fishes have not been investigated. Therefore, in this study, we cloned and characterized zebrafish CDKL5 (zCDKL5) and its substrate, amphiphysin 1 (zAmph1).

The Phosphatase-Resistant Isoform of CaMKI, Ca²⁺/Calmodulin-Dependent Protein Kinase Iδ (CaMKIδ), Remains in Its "Primed" Form without Ca²⁺ Stimulation.

Ca²⁺/calmodulin-dependent protein kinase I (CaMKI) is known to play pivotal roles in Ca²⁺ signaling pathways. Four isoforms of CaMKI (α, β, γ, and δ) have been reported so far. CaMKI is activated through phosphorylation by the upstream kinase, CaMK kinase (CaMKK), and phosphorylates downstream targets.

TandeMBP: generation of a unique protein substrate for protein kinase assays.

Myelin basic protein (MBP) is one of the major components of central nervous system myelin and has multiple sites for protein phosphorylation. Therefore, it has been widely used as a substrate for in vitro assays of various protein kinases. In this study, to obtain more efficient substrates for protein kinase assays than commercially available MBP from bovine brain, we produced various recombinant MBPs using Escherichia coli expression systems.

Expression and gene knockdown of zebrafish Ca(2+)/calmodulin-dependent protein kinase Iδ-LL.

Ca(2+)/calmodulin-dependent protein kinase Iδ (CaMKIδ) is expressed ubiquitously, but little is known about its physiological functions. Recently, we cloned and characterized two splice variants of zebrafish (Danio rerio) CaMKIδ (CaMKIδ-S/L). In the present study we cloned a new CaMKIδ isoform, CaMKIδ-LL, encoded by a different gene from CaMKIδ-S/L.

Knockdown of two splice variants of Ca(2+)/calmodulin-dependent protein kinase Iδ causes developmental abnormalities in zebrafish, Danio rerio.

We isolated cDNA clones for zebrafish Ca(2+)/calmodulin-dependent protein kinase I (zCaMKI) δ isoforms by expression screening using cDNA library from embryos at 72-h post-fertilization (hpf). There are two splice variants with different C-terminal sequences, comprising of 392 and 368 amino acids, and they are designated zCaMKIδ-L (long form) and zCaMKIδ-S (short form), respectively. Although recombinant zCaMKIδ-L and zCaMKIδ-S expressed in Escherichia coli showed essentially the same catalytic properties including substrate specificities, they showed different spatial and temporal expression.

Detection of protein kinase substrates in tissue extracts after separation by isoelectric focusing.

Here we report a simple and useful method to detect endogenous substrates of protein kinases. When crude tissue extracts were resolved by liquid-phase isoelectric focusing (MicroRotofor) and the separated protein fractions were phosphorylated by protein kinases such as Ca(2+)/calmodulin-dependent protein kinase I or cAMP-dependent protein kinase, various proteins in the different fractions were efficiently phosphorylated. Since a higher number of substrates could significantly be detected using the resolved fractions by MicroRotofor as compared to direct analysis of the original tissue extracts, our present method will be applicable to the screening of endogenous substrates for various protein kinases.