Transformation strategies for stable expression of complex hetero‐multimeric proteins like secretory immunoglobulin A in plants

Plant expression systems have proven to be exceptional in producing high‐value complex polymeric proteins such as secretory IgAs (SIgAs). However, polymeric protein production requires the expression of multiple genes, which can be transformed as single or multiple T‐DNA units to generate stable transgenic plant lines. Here, we evaluated four strategies to stably transform multiple genes and to obtain high expression of all components. Using the in‐seed expression of a simplified secretory IgA (sSIgA) as a reference molecule, we conclude that it is better to spread the genes over two T‐DNAs than to contain them in a single T‐DNA, because of the presence of homologous recombination events and gene silencing. These T‐DNAs can be cotransformed to obtain transgenic plants in one transformation step. However, if time permits, more transformants with high production levels of the polymeric protein can be obtained either by sequential transformation or by in‐parallel transformation followed by crossing of transformants independently selected for excellent expression of the genes in each T‐DNA.     

Modulating secretory pathway pH by proton channel co‐expression can increase recombinant protein stability in plants

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host‐cell engineering approaches are being developed to ameliorate host‐cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid‐susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co‐expression. The co‐expression of a heterologous ion channel to stabilize acid‐labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.     

A large‐scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand‐binding domains of taste receptor

Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo‐ or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co‐expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large‐scale expression of hetero‐multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high‐expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large‐scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY‐2) suspension cells

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease‐deficient tobacco BY‐2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY‐2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full‐length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV‐1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four‐fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N‐terminal sequencing data revealed that the antibody has two cleavage sites within the CDR‐H3 and one site at the end of the H4‐framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.     

mRNA/lncRNA expression patterns and the function of fibrinogen‐like protein 2 in Meishan pig endometrium during the preimplantation phases

Embryonic implantation involves a complex and well‐coordinated interaction between the developing conceptus and maternal uterus, and the preimplantation period has a major impact on litter size in pigs. The present study aimed to investigate the vital messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) that regulate preimplantation in Meishan pigs. The enriched Gene Ontology terms were all related to “binding.” Furthermore, “ECM‐receptor interaction” was predicted as an important pathway that regulated the success of implantation. We speculated that the differentially expressed mRNAs S100A9, ANXA8, MUC16, and FGL2 and the differentially expressed lncRNAs TCONS_11206566, TCONS_09904861, and TCONS_1252933 may play vital roles in the process of implantation. Furthermore, this study verified that FGL2 was highly expressed on Day 12 of pregnancy, and we also investigated the function of FGL2 during preimplantation in vivo. In conclusion, this study provides useful information for further analyses of the molecular mechanisms of implantation in Chinese domestic pigs.     

A study of the possible association between adenosine A2A receptor gene polymorphisms and attention‐deficit hyperactivity disorder traits

The adenosine A_2A receptor (ADORA2A) is linked to the dopamine neurotransmitter system and is also implicated in the regulation of alertness, suggesting a potential association with attention‐deficit hyperactivity disorder (ADHD) traits. Furthermore, animal studies suggest that the ADORA2A may influence ADHD‐like behavior. For that reason, the ADORA2A gene emerges as a promising candidate for studying the etiology of ADHD traits. The aim of this study was to examine the relationship between ADORA2A gene polymorphisms and ADHD traits in a large population‐based sample. This study was based on the Child and Adolescent Twin Study in Sweden (CATSS), and included 1747 twins. Attention‐deficit hyperactivity disorder traits were assessed through parental reports, and samples of DNA were collected. Associations between six single nucleotide polymorphisms (SNPs) and ADHD traits were examined, and results suggested a nominal association between ADHD traits and three of these SNPs: rs3761422, rs5751876 and rs35320474. For one of the SNPs, rs35320474, results remained significant after correction for multiple comparisons. These results indicate the possibility that the ADORA2A gene may be involved in ADHD traits. However, more studies replicating the present results are warranted before this association can be confirmed .     

Association between high‐sensitivity C‐reactive protein,lipoprotein‐associated phospholipase A2 and carotid atherosclerosis: A cross‐sectional study

High‐sensitivity C‐reactive protein (hs‐CRP) and lipoprotein‐associated phospholipase A2 (Lp‐PLA2) have been reported to be independent predictors of atherosclerosis. However, whether the combination of these two markers can improve the prediction of atherosclerosis is unknown. This study aimed to evaluate the association between combining hs‐CRP and Lp‐PLA2 and predicting carotid atherosclerosis. A total of 1982 participants aged ≥40 years were included in this study. Hs‐CRP and Lp‐PLA2 were measured by a high‐sensitivity nephelometry assay and quantitative sandwich enzyme‐linked immunosorbent assay, respectively. Ultrasonography was performed on the bilateral carotid arteries to evaluate stenosis and plaques. Multivariable logistic regression models were used to analyse the association between the combination of the hs‐CRP and Lp‐PLA2 levels and carotid plaques and stenosis. A total of 1579 (79.7%) and 181 (9.1%) subjects had carotid plaques and carotid stenosis, respectively. The group with high hs‐CRP and Lp‐PLA2 levels had the highest prevalence of carotid plaques (90.6%) and stenosis (20.8%). A significant association was found between high hs‐CRP and Lp‐PLA2 levels and carotid stenosis (adjusted odds ratio [OR]: 2.39; 95% confidence interval [CI]: 1.13‐5.09), but this combination was not associated with carotid plaques (OR: 2.62, 95% CI: 0.93‐7.38). The results suggested that the combination of hs‐CRP and Lp‐PLA2 were better predictors than either protein alone with regard to carotid atherosclerosis.     

Neither single‐marker nor haplotype analyses support an association between the dopamine transporter gene and heroin dependence in Han Chinese

Much evidence suggests that dysfunction of dopamine transporter‐mediated dopamine transmission may be involved in the pathophysiology of substance abuse and dependence. The aim of this study was to examine whether the dopamine transporter gene (DAT1; SLC6A3) is associated with the development of heroin dependence (HD) and whether DAT1 influences personality traits in patients with HD. Polymorphisms of DAT1 were analyzed in a case–control study of 1046 Han Chinese (615 patients and 431 controls). All participants were screened using a Chinese version of the modified Schedule of Affective Disorder and Schizophrenia‐Lifetime and all patients met the criteria for HD. Furthermore, a Chinese version of the Tridimensional Personality Questionnaire (TPQ) was used to assess personality traits in the patient group and examine the association between their personality traits and DAT1 polymorphisms. Of the patient group, 271 completed the TPQ. No statistically significant differences in allele or genotype frequencies of all investigated variants between HD patients and controls were observed. In haplotype analyses, four haplotype blocks of DAT1 were not associated with the development of HD. These DAT1 polymorphisms did not influence novelty seeking and harm avoidance scores in HD patients. This study suggests that the DAT1 gene may not contribute to the risk of HD and specific personality traits in HD among the Han Chinese population.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.