Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

Background: Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector® micro-fermentation system. Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl -D-thiogalactopyranoside) and IPTG (isopropyl -D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). Conclusions: We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.(VLID)101641

Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Microbial Cell Factories / Tuning constitutive recombinant gene expression in Lactobacillus plantarum

Background: Lactobacillus plantarum constitutes a well-recognized food-grade system for the expression of recombinant proteins in the field of industrial and medical biotechnology. For applications in vivo or in biotechnological processes, the level of expression of e.g. antigens or enzymes is often critical, as expression levels should be of a certain effectiveness, yet, without putting too much strain to the overall system. The key factors that control gene expression are promoter strength, gene copy number and translation efficiency. In order to estimate the impact of these adjusting screws in L. plantarum CD033, we have tested several constitutive promoters in combination with high and low copy number plasmid backbones and varying space between the Shine-Dalgarno sequence and the start-codon. Results: By combining strong promoters, such as transcription elongation factor promoters, isolated from L. plantarum CD033 and L. buchneri CD034, a synthetic promoter, originally derived from L. plantarum WCSF1 and a heterologous promoter derived from L. buchneri CD034 with a high and a low copy number origin of replication we demonstrated various expression levels of the model protein mCherry. All promoters were feasible for protein expression and in all cases, the high copy number origin of replication increased expression twofold. We found that the optimal spacer between the Shine-Dalgarno sequence and the start codon in L. plantarum consists of 8 nucleotides and elongation as well as shortening this sequence gradually down-regulates gene expression. Conclusions: We have evaluated the effects of a set of gene regulatory tools to fine tune recombinant gene expression in L. plantarum CD033. We have thus, provided potential expression vectors useful for constitutive protein expression in lactic acid bacteria ranging from moderate to strong production levels

Teasing, taunting, and the politics of politeness: high sociometric status is associated with expectation-consistent behavior.

Research examining face-to-face status hierarchies suggests that individuals attain respect and admiration by engaging in behavior that influences others' judgments of their value to the group. Building on this research, we expected that high-status individuals would be less likely to engage in behaviors that violate group norms and expectations, relative to low-status individuals. Adolescent participants took part in an interaction in which they teased an opposite-gender friend (Study 1) or an experiment in which taunting or cheering expectations were manipulated (Study 2). Consistent with the hypothesis, high-status boys and girls engaged in teasing behaviors consistent with their gender roles, relative to their low status counterparts (Study 1). In Study 2, high-status boys engaged in more direct provocation and off-record commentary while taunting, and more affiliative behavior while cheering on their partner, relative to low-status boys. Discussion focused on how expectation-consistent actions help individuals maintain elevated status

Teasing, Taunting, and the Politics of Politeness: High Sociometric Status Is Associated with Expectation-Consistent Behavior

<div><p>Research examining face-to-face status hierarchies suggests that individuals attain respect and admiration by engaging in behavior that influences others' judgments of their value to the group. Building on this research, we expected that high-status individuals would be less likely to engage in behaviors that violate group norms and expectations, relative to low-status individuals. Adolescent participants took part in an interaction in which they teased an opposite-gender friend (Study 1) or an experiment in which taunting or cheering expectations were manipulated (Study 2). Consistent with the hypothesis, high-status boys and girls engaged in teasing behaviors consistent with their gender roles, relative to their low status counterparts (Study 1). In Study 2, high-status boys engaged in more direct provocation and off-record commentary while taunting, and more affiliative behavior while cheering on their partner, relative to low-status boys. Discussion focused on how expectation-consistent actions help individuals maintain elevated status.</p></div

Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2Azk variants were efficiently produced in fed-batch like μ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications

Plasmids and plasmid amounts used for various experiments.

<p>pEGFP-N1, pcDNA luc, pRC-LC and pRC-HC carry the CMV immediate-early promoter, pGL3-control and pRL carry SV40 promoter elements, pTripolis and pBi-NP drive bidirectional transcription (mRNA and vRNA) of the influenza polymerases and the NP protein. pMono-EGFP, pMono-lucNS, pMono-LC, pMono-HC, pMono-lucM and pMono-lucPB1 drive monodirectional (vRNA) transcription of the gene of interest. AB stands for antibody, LC stands for light chain experiments.</p

Expression of a the monoclonal anti-gp41 antibody 3D6.

<p>HEK-293 cells were either tranfected with pRC-LC and pRC-HC (CMV HC∶LC = 1∶1, CMV HC∶LC = 10∶1) or with the influenza replicon and pMono-LC and pMono-HC (Flu Replicon HC∶LC = 1∶1, Flu Replicon HC∶LC = 10∶1). Heavy chain to light chain ratios of 1∶1 and 10∶1 were tested. Antibody concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.</p