Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required

A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein

10.1371/journal.pone.0033451PLoS ONE74

Dominância fiscal : uma investigação empírica sobre o caso brasileiro no período de 2003 a 2014

A estabilização econômica dos anos de 1990 e a adoção do tripé econômico, a partir de 1999, marcam o fim de um capítulo delicado da história brasileira; a partir de então, era necessária a existência de certa sintonia de políticas monetária e fiscal para a manutenção do controle dos diversos indicadores econômicos. Contudo, com essa reciprocidade na política econômica, são incitadas discussões sobre a orientação do governo na hora de definir suas prioridades nesse campo: as variáveis fiscais são priorizadas e, por conseguinte, determinadas, forçando as monetárias a se ajustarem – ou o contrário? A resposta para esse questionamento leva à discussão sobre a dominância fiscal. Assim, esse trabalho visa verificar empiricamente, usando das modelagens econométricas VAR e estudo de eventos, se há dominância fiscal ou monetária na economia brasileira e se a eficácia da política monetária mudou na transição do governo Lula para o governo Dilma. O resultado foi inconclusivo para o governo Lula e indicou dominância fiscal no governo Dilma. Ainda verificou-se não haver modificação na eficácia da política monetária.Economic stabilization, in the 1990s, and utilization of an economic tripod, after 1999, represents the end of a delicate chapter in Brazilian history. Ever since, it was necessary the existence of a certain agreement between monetary and fiscal politic, in order to maintain under control a variety of economic indicators. However, this reciprocity (in economic politic) starts discussions about the real government orientations when it comes to define its priority on this subject: are the fiscal variables priorized, and then, determined, forcing monetary variables to adjust themselves, or the opposite? The answer to these questions emerge from the fiscal dominance discussion. This paper intends to empiric verify, using econometric modeling VAR and event study, if there is fiscal dominance or monetary in Brazilian economy and whether the effectiveness of monetary politic has changed in the transition from Lula's government to the Dilma government. The result was inconclusive for the Lula government and indicated fiscal dominance in the Dilma government. There was still no change in the efficiency of the monetary politic.CAPE

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Comparison between bacterial and mammalian IgG.

<p>(A). Non-reducing SDS-PAGE of 2 µg of pooled bacterial IgG eluate after protein L purification (EL) and mammalian cell culture-derived IgG (IgG) indicates that the majority of purified IgG was fully assembled IgG (2H2L) although partially assembled IgG (2H1L, 1H1L) are also present. (B). Direct binding ELISA of serially diluted bacterial (-▪-)- and mammalian cell culture (-•-)-derived IgG against Dengue serotype-2 virus and epsilon toxin showing similar binding curves. Binding of antibody was detected using anti-Human IgG Fc-HRPO as the secondary antibody.</p

Large scale purification of bacterial IgG.

<p>(A) Non-reducing coomassie gel of peak fractions from Protein A HPLC purification of cell lysate. Left panel: 4G2 fractions 5 to 11 (F5 to F11). Right panel ET149 fractions 10 to 16 (F10 to F16). 30 µl of each fraction was run on the gel. A sample of the wash (LW) was run and shows no contaminants present indicating the column was sufficiently washed to remove non-binding proteins. Fully assembled IgGs (2H2L) are indicated. (B). Reducing coomassie gels (4G2-F7, ET149-F12 & 13) and adjacent immunoblots (I) showing representative fractions from each Protein A elution (indicated with * on panel A). 30 µl of each fraction was loaded for coomassie and 3.75 µl for immunoblot. Blots were probed with both anti-IgG Fc and anti-Kappa chain polyclonals showing that majority of protein bands in the fraction are neither IgG heavy chain nor light chain. A separate reducing coomassie gel shows 2 µg of bacterial IgG pooled eluate (EL) after Protein L purification showing successful removal of the contaminating proteins and degraded heavy chain fragments. The equivalent amount of mammalian cell culture-derived IgG (IgG) was loaded for comparison. Individual heavy (HC) and light (LC) chains are indicated although the light chain for ET149 appears as two separate species.</p

Initial bacterial IgG expression and periplasmic extraction.

<p>(A and B) Non-reducing and reducing immunoblot of periplasmic extract from overnight expression of 4G2 in HB2151 (HB) and BL21(DE3) (BL) <i>E. coli</i> strains, respectively. All blots were probed with anti-Human Fc-HRPO.</p

Small scale optimization of bacterial IgG expression.

<p>(A-E) Left and middle panels: Non-reducing immunoblot of 7.5 µl clarified cell lysate from small scale overnight expression of bacterial IgG in shaking culture. All blots were probed with anti-Human Fc-HRPO and adjusted to ensure equal intensity. Arrows indicate fully-assembled IgG. Right panel: Direct binding ELISA indicating levels of functional IgG. Background binding signal was negligible for all cell lysate samples at the indicated dilution or neat. Error bars were calculated from average of three or four observations. Expressed antibodies were 4G2 (A), PA38 (B), PA64 (C), ET21 (D), ET149 (E). (F) Variation in wet cell mass for all five antibodies under different induction conditions. Mass is indicated in mg</p

Construction of tet promoter bacterial IgG expression plasmid.

<p>pASK-IBA2 plasmid using a <i>tet</i> promoter was used as the backbone expression vector. The appropriate restriction sites for cloning in of the light and variable heavy chains, leader sequences (OmpA, PelB) and the constant heavy chain sequence (CH) were added. Light (LC) and variable chains (VH) were cloned in as a complete construct together with the intercistronic ribosomal binding site (RBS) from the phage display vector or as separate constructs from the 4G2 mammalian IgG expression vector.</p