Determination of the optimal connector length to enhance stability of backbone-circularized granulocyte colony-stimulating factor.

Improving protein stability is important for industrial applications, and one promising method for achieving this is backbone circularization. As connector length affects stability, predicting and elucidating a more stable connector length is necessary for development of the backbone circularization method. However, the relationship between connector length and protein stability has not been completely elucidated. Here, we determined the most stable connector length for granulocyte colony-stimulating factor by changing one residue at a time to produce connector length variants and then measuring their thermal stability. Analysis of the local structures obtained from the predicted structures of the circularized variants revealed that an approach using helix length, dihedral backbone angle, and number of unbonded hydrogen bond donors and acceptors is suitable for identifying connector lengths with higher stability.

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.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.

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. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.

Autoactivation of C-terminally truncated Ca2+/calmodulin-dependent protein kinase (CaMK) Iδ via CaMK kinase-independent autophosphorylation.

Ca2+/calmodulin-dependent protein kinase I isoforms (CaMKIα, ß, γ, and δ) play important roles in Ca2+ 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. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1-299) and rCaMKIα(1-294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1-299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1-299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1-294) was not. The major autophosphorylation site leading to activation of the kinase was Ser296, determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser296 in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser296, which might be a clue to understand the physiological regulation of CaMKIδ isoform.

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 this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.

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. Here, we introduce a scanning electron-assisted dielectric microscopy (SE-ADM) technique for in-solution imaging of monoclonal immunoglobulin G (IgG) aggregates without staining and drying. Remarkably, SE-ADM allowed assessment of the size and morphology of the IgG aggregates in solution by completely excluding drying-induced artifacts. SE-ADM was also beneficial to study IgG aggregation caused by temporary acid exposure followed by neutralization, pH-shift stress. A box-counting analysis of the SE-ADM images provided fractal dimensions of the larger aggregates, which complemented the fractal dimensions of the smaller aggregates measured by light scattering. The scale-free or self-similarity nature of the fractal aggregates indicated that a common mechanism for antibody aggregation existed between the smaller and larger aggregates. Consequently, SE-ADM is a useful method for characterizing protein aggregates to bridge the gaps that occur among conventional analytical methods, such as those related to in situ/ ex situ techniques or size/morphology assessments.

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, 0.22-µm membrane filters are commonly used to remove aggregates. However, particles with a diameter below 0.22 µm (small aggregates) are not removed from the final product. The residual species may grow into large aggregates during the storage period. In the current study, we devised a strategy to suppress IgG aggregate growth by removing aggregation precursors using the artificial protein AF.2A1. This protein efficiently binds the Fc region of non-native IgG conformers generated under chemical and physical stresses. Magnetic beads conjugated with AF.2A1 were used to remove non-native monomers and aggregates from solutions of native IgG and from native IgG solutions spiked with stressed IgG. The time-dependent growth of aggregates after the removal treatment was monitored. The removal of aggregation precursors, i.e., non-native monomers and nanometer aggregates (<100 nm), suppressed the aggregate growth. The presented findings demonstrate that a removal treatment with a specific adsorbent of non-native IgG conformers enables long-term stable storage of therapeutic IgG molecules and will facilitate mitigation of the immunogenicity of IgG preparations.

Characterization of CoPK02, a Ca2+/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. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca2+/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca2+/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca2+-signaling in C. cinerea.

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. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing 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. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.

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. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.

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). Two alternative splice variants of zCDKL5, zCDKL5-Long (zCDKL5-L) and zCDKL5-Short (zCDKL5-S), and three splice variants of zAmph1, zAmph1a, zAmph1b and zAmph1c, were cloned from a zebrafish cDNA library. Using zAmph1a point mutants, we identified Ser-285 and Ser-293 as phosphorylation sites of zAmph1a by CDKL5. Transiently expressed zCDKL5-L and zCDKL5-S colocalized with zAmph1a in the cytoplasm of 293T cells. RT-PCR analysis revealed that zCDKL5-L was first observed 12hours post-fertilization (hpf) and increased thereafter, while zCDKL5-S appeared just after fertilization. zAmph1a was detected in all embryogenic stages and zAmph1b appeared from 12hpf, but the expression of zAmph1c was not observed in our experiments. In adult fish, zCDKL5-L was mainly expressed in the brain, but zCDKL5-S showed ubiquitous expression. zAmph1a was observed most abundantly in the eyes, whereas zAmph1b was predominantly expressed in the brain. zAmph1c was scarcely detected. These results suggest that phosphorylation of Amph1 by CDKL5 may be a common feature throughout animal species.

The Phosphatase-Resistant Isoform of CaMKI, Ca²âº/Calmodulin-Dependent Protein Kinase Iδ (CaMKIδ), Remains in Its "Primed" Form without Ca²âº Stimulation.

Ca²âº/calmodulin-dependent protein kinase I (CaMKI) is known to play pivotal roles in Ca²âº signaling pathways. Four isoforms of CaMKI (α, ß, γ, and δ) have been reported so far. CaMKI is activated through phosphorylation by the upstream kinase, CaMK kinase (CaMKK), and phosphorylates downstream targets. When CaMKI was transiently expressed in 293T cells, CaMKIα was not phosphorylated at all under low-Ca²âº conditions in the cells. In contrast, we found that CaMKIδ was significantly phosphorylated and activated to phosphorylate cAMP response element-binding protein (CREB) under the same conditions. Herein, we report that the sustained activation of CaMKIδ is ascribed to its phosphatase resistance resulting from the structure of its N-terminal region. First, we examined whether CaMKIδ is more readily phosphorylated by CaMKK than CaMKIα, but no significant difference was observed. Next, to compare the phosphatase resistance between CaMKIα and CaMKIδ, we assessed the dephosphorylation of the phosphorylated CaMKIs by CaMK phosphatase (CaMKP/PPM1F). Surprisingly, CaMKIδ was hardly dephosphorylated by CaMKP, whereas CaMKIα was significantly dephosphorylated under the same conditions. To date, there have been no detailed reports concerning dephosphorylation of CaMKI. Through extensive analysis of CaMKP-catalyzed dephosphorylation of various chimeric and point mutants of CaMKIδ and CaMKIα, we identified the amino acid residues responsible for the phosphatase resistance of CaMKIδ (Pro-57, Lys-62, Ser-66, Ile-68, and Arg-76). These results also indicate that the phosphatase resistance of CaMKI is largely affected by only several amino acids in its N-terminal region. The phosphatase-resistant CaMKI isoform may play a physiological role under low-Ca²âº conditions in the cells.

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. Three splice isoforms of mouse MBP were expressed in E. coli and successfully purified using a new protocol consisting of HCl extraction, urea treatment and affinity purification with HiTrap Chelating HP column. The recombinant MBP isoforms thus obtained served as more efficient substrates for protein kinases than MBP isolated from bovine brain. To generate an even better substrate for protein kinase assays, we produced a hybrid protein composed of two different MBP isoforms connected in tandem, designated TandeMBP. TandeMBP was readily expressed in E. coli and could be purified by the newly developed simple procedure. TandeMBP was phosphorylated by various Ser/Thr protein kinases more efficiently than the other MBP isoforms. Taken together, TandeMBP will become a powerful tool for in vitro assays to analyse various protein kinase activities.

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. While the catalytic domain of CaMKIδ-LL showed 86% identity that of CaMKIδ-S/L, it had a unique C-terminal sequence. To clarify the functional role of CaMKIδ-LL, we investigated the biological significance of this new isoform during zebrafish embryogenesis. Although CaMKIδ-LL exhibited essentially the same catalytic properties and substrate specificities as the other CaMKIδ isoforms, it showed different temporal and spatial expression. During zebrafish embryogenesis, RT-PCR analysis detected CaMKIδ-LL expression after 48 h post-fertilization. Western blotting in adult zebrafish demonstrated that CaMKIδ-LL is expressed in the brain, the eye, and, abundantly, in fins. Knockdown of CaMKIδ-LL expression using morpholino-based antisense oligonucleotides resulted in an increase in abnormal embryos with small fins and underdeveloped cartilage. These phenotypes were rescued by co-injection with recombinant CaMKIδ-LL. These results clearly indicated that CaMKIδ-LL plays an important role in the generation of cartilage and fins during zebrafish embryogenesis.

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. Western blotting analysis using the isoform-specific antibodies revealed that zCaMKIδ-L clearly appeared from 36hpf but zCaMKIδ-S began to appear at 60hpf and thereafter. zCaMKIδ-S was predominantly expressed in brain, while zCaMKIδ-L was widely distributed in brain, eye, ovary and especially abundantly expressed in skeletal muscle. The gene knockdown of zCaMKIδ using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. Severe phenotype of embryos exhibited short trunk, kinked tail and small heads. These phenotypes could be rescued by coinjection with the recombinant zCaMKIδ, but not with the kinase-dead mutant. These results clearly indicate that the kinase activity of zCaMKIδ plays a crucial role in the early stages in the embryogenesis of zebrafish.

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.