Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Identification of candidate chemosensory receptors in the antennal transcriptome of Tropidothorax elegans

Molecular Biology Reports - Chemosensory receptors in the dendritic membrane of olfactory cells are critical for the molecular recognition and discrimination of odorants. Tropidothorax elegans is a...     

Longitudinal virological changes and underlying pathogenesis in hospitalized COVID-19 patients in Guangzhou,China

Science China Life Sciences - Prolonged viral RNA shedding and recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in coronavirus disease 2019 (COVID-19) patients have been...     

Protective Effects of Pidotimod Against Salmonella Infections

International Journal of Peptide Research and Therapeutics - Pidotimod has been shown to exhibit immunomodulatory activities and exert protective effects against bacterial infections. This study...     

Establishment of a micropropagation supporting technology for the Fraxinus mandshurica × Fraxinus sogdiana

In Vitro Cellular & Developmental Biology - Plant - The interspecific hybridization can take advantage of heterosis and combine the double parental traits most extensively, and is an effective...     

Evidence for a synergistic effect of post‐translational modifications and genomic composition of eEF‐1α on the adaptation of Phytophthora infestans

Genetic variation plays a fundamental role in pathogen''s adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post‐translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor‐1α (eEF‐lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF‐lα protein, possibly attributable to purifying selection and a lack of intra‐genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF‐1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF‐1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre‐ and post‐translational phase in eEF‐1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。     

Bone morphogenetic protein 9 overexpression reduces osteosarcoma cell migration and invasion

Transforming growth factor-β (TGF-β) is known to promote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF-β family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the expression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.