Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

Concomitant reduction of lactate and ammonia accumulation in fed‐batch cultures: Impact on glycoprotein production and quality

Lactate and ammonia accumulation is a major factor limiting the performance of fed‐batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by‐products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine‐free fed‐batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2‐overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed‐batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine‐free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 and 1 mM, respectively. In flask, this resulted in more than a 2.5‐fold increase in IFNα2b titer in comparison to the control glutamine‐supplied fed‐batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC‐MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high‐quality therapeutic glycoproteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:494–504, 2018     

Identification of the interaction between bta‐miR‐370 and OLR1 gene in bovine adipocyte

It has been shown that the oxidized low density lipoprotein receptor 1 (OLR1) gene plays an important role in the degradation of oxidized low density lipoprotein. Previous studies found a SNP in the 3′‐untranslated region (3′‐UTR) of the OLR1 gene associated with milk production traits in different dairy cattle populations and with loin eye area and marbling depth in beef cattle. MicroRNAs can regulate gene expression by binding the 3′‐UTR of target genes to degrade or to repress the translation of target genes. Bioinformatics have shown that there is a binding site of bta‐miR‐370 in the 3′‐UTR of the OLR1 gene, and a previous luciferase reporter assay system showed that the A/C mutation occurring in the 3′‐UTR of this gene caused the binding sites of bta‐miR‐370 to disappear in HEK293 cells. To further validate whether OLR1 was the target gene of bta‐miR‐370, the over‐expression and interference expression of bta‐miR‐370 were determined by transfecting bta‐miR‐370 mimics and inhibitor supplementations into bovine adipocyte. The qRT‐PCR result showed that the relative expression of OLR1 gene significantly decreased in the mimics group compared to the control, whereas the expression level in inhibitor group was higher than its control group. The above results were further verified by a Western blot at the protein level. In addition, lipid formation analysis of bovine adipocytes was performed via oil red O staining, and we found that cytoplasm lipid droplets in the inhibitor group showed a tendency to increase compared to the control group, whereas in the mimics group, we observed an obvious decrease of cytoplasm lipid droplets compared to the control and inhibitor groups. Taken together, our data here suggest that bta‐miR‐370 has a negative regulation role for OLR1 both at the gene expression and protein levels and bovine adipocytes cytoplasm lipid droplets formation, which provides a reference for illustrating how the OLR1 gene affects milk production and beef quality traits in cattle.     

AP2α is essential for MUC8 gene expression in human airway epithelial cells

Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.     

A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells 1 but reported yields from Chinese hamster ovary (CHO) cells are lower at ～300 mg/L. 2 We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X‐box binding protein (XBP‐1S) and endoplasmic reticulum oxidoreductase (ERO1‐Lα) and has been named CHOS‐XE. CHOS‐XE cells produced increased antibody (MAb) yields (5.3– 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:697–706, 2013     

The effect of Bcl‐2, YAMA,and XIAP over‐expression on apoptosis and adenovirus production in HEK293 cell line

Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over‐expression of anti‐apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large‐scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type‐5‐adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over‐expression of bcl‐2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over‐expression of pro‐caspase‐3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase‐3 dependent apoptosis, and to a lesser degree caspase‐9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over‐expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase‐mediated activation plays an important role in virus infection and maturation. Biotechnol. Bioeng. 2009; 104: 752–765 © 2009 Wiley Periodicals, Inc.     

Characterization and high‐yield production of non‐N‐glycosylated recombinant human BCMA‐Fc in Pichia pastoris

B‐cell maturation antigen (BCMA) fused at the C‐terminus to the Fc portion of human IgG1 (BCMA‐Fc) blocks B‐cell activating factor (BAFF) and proliferation‐inducing ligand (APRIL)‐mediated B‐cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA‐Fc (rhBCMA‐Fc) in Pichia pastoris under the control of the AOX1 methanol‐inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N‐linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed‐batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA‐Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA‐Fc possessed high‐binding affinity to APRIL and dose‐dependent inhibition of APRIL‐induced proliferative activity in vitro through three‐step purification. Thus, this yeast‐derived expression method could be a low‐cost and effective alternative to the production of rhBCMA‐Fc in mammalian cell lines.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.     

Competing G protein‐coupled receptor kinases balance G protein and β‐arrestin signaling

Seven‐transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β‐arrestins, whose recruitment to the activated receptor is regulated by G protein‐coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal‐regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT_1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)‐based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well‐established function in the desensitization of G‐protein activation, GRK2 exerts a strong negative effect on β‐arrestin‐dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2‐dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT_1AR, and HEK293 cells expressing other 7TMRs.     

Runx2‐mediated bcl‐2 gene expression contributes to nitric oxide protection against hydrogen peroxide‐induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. This study was aimed to evaluate the protective effects of pretreatment with sodium nitroprusside (SNP) as a source of NO on hydrogen peroxide‐induced osteoblast insults and its possible mechanisms. Exposure of human osteosarcoma MG63 cells to hydrogen peroxide significantly increased cellular oxidative stress, but decreased ALP activity and cell viability, inducing cell apoptosis. Pretreatment with 0.3 mM SNP significantly lowered hydrogen peroxide‐induced cell insults. Treatment of human MG63 cells with hydrogen peroxide inhibited Bcl‐2 mRNA and protein production, but pretreatment with 0.3 mM SNP significantly ameliorated such inhibition. Sequentially, hydrogen peroxide decreased the mitochondrial membrane potential, but increased the levels of cytochrome c and caspase‐3 activity. Pretreatment with 0.3 mM SNP significantly lowered such alterations. Exposure to hydrogen peroxide decreased Runx2 mRNA and protein syntheses. However, pretreatment with 0.3 mM SNP significantly lowered the suppressive effects. Runx2 knockdown using RNA interference inhibited Bcl‐2 mRNA production in human MG63 cells. Protection of pretreatment with 0.3 mM SNP against hydrogen peroxide‐induced alterations in ALP activity, caspase‐3 activity, apoptotic cells, and cell viability were also alleviated after administration of Runx2 small interference RNA. Thus, this study shows that pretreatment with 0.3 mM SNP can protect human MG63 cells from hydrogen peroxide‐induced apoptotic insults possibly via Runx2‐involved regulation of bcl‐2 gene expression. J. Cell. Biochem. 108: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver‐directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni²⁺ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni²⁺ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.