Characterization of the mutant lytic state in lambda expression systems

AbstractThe development of therapeutic proteins for the treatment of numerous diseases is one of the fastest growing areas of biotechnology. Therapeutic efficacy and serum half-life are particularly important, and these properties rely heavily on the glycosylation state of the protein. Expression systems to produce authentically fully glycosylated therapeutic proteins with appropriate terminal sialic acids are not yet perfected. The in vitro modification of therapeutic proteins by recombinant sialyltransferases offers a promising and elegant strategy to overcome this problem. Thus, the detailed expression and characterization of sialyltransferases for completion of the glycan chains is of great interest to the community. We identified a novel α2,6-sialyltransferase from Helicobacter cetorum and compared it to the human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. We demonstrated that the recombinant α2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.

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Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.00365-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3802-3812 ◽

Cited By ~ 45

Author(s):

Juergen Mairhofer ◽

Theresa Scharl ◽

Karoline Marisch ◽

Monika Cserjan-Puschmann ◽

Gerald Striedner

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Gene Dosage ◽

Expression System ◽

Transcriptional Level ◽

Strong Impact ◽

Expression Systems ◽

Free System ◽

Recombinant Gene Expression

ABSTRACTPlasmid-basedEscherichia coliBL21(DE3) expression systems are extensively used for the production of recombinant proteins. However, the combination of a high gene dosage with strong promoters exerts extremely stressful conditions on producing cells, resulting in a multitude of protective reactions and malfunctions in the host cell with a strong impact on yield and quality of the product. Here, we provide in-depth characterization of plasmid-based perturbations in recombinant protein production. A plasmid-free T7 system with a single copy of the gene of interest (GOI) integrated into the genome was used as a reference. Transcriptomics in combination with a variety of process analytics were used to characterize and compare a plasmid-free T7-based expression system to a conventional pET-plasmid-based expression system, with both expressing human superoxide dismutase in fed-batch cultivations. The plasmid-free system showed a moderate stress response on the transcriptional level, with only minor effects on cell growth. In contrast to this finding, comprehensive changes on the transcriptome level were observed in the plasmid-based expression system and cell growth was heavily impaired by recombinant gene expression. Additionally, we found that the T7 terminator is not a sufficient termination signal. Overall, this work reveals that the major metabolic burden in plasmid-based systems is caused at the level of transcription as a result of overtranscription of the multicopy product gene and transcriptional read-through of T7 RNA polymerase. We therefore conclude that the presence of high levels of extrinsic mRNAs, competing for the limited number of ribosomes, leads to the significantly reduced translation of intrinsic mRNAs.

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An Interscholastic Network to Generate LexA Enhancer Trap Lines in Drosophila

10.1101/552513 ◽

2019 ◽

Author(s):

Lutz Kockel ◽

Catherine Griffin ◽

Yaseen Ahmed ◽

Lauren Fidelak ◽

Arjun Rajan ◽

...

Keyword(s):

Molecular Basis ◽

Tissue Expression ◽

Enhancer Trap ◽

Source Distribution ◽

Expression Systems ◽

Tissue Expression Analysis ◽

Experimental Tool ◽

Chromosome Iii ◽

Enhancer Trap Lines

AbstractBinary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StonEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

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False Biomarker Discovery due to Reactivity of a Commercial ELISA for CUZD1 with Cancer Antigen CA125

Clinical Chemistry ◽

10.1373/clinchem.2013.215236 ◽

2014 ◽

Vol 60 (2) ◽

pp. 381-388 ◽

Cited By ~ 28

Author(s):

Ioannis Prassas ◽

Davor Brinc ◽

Sofia Farkona ◽

Felix Leung ◽

Apostolos Dimitromanolakis ◽

...

Keyword(s):

Biomarker Discovery ◽

Size Exclusion ◽

Ductal Adenocarcinoma ◽

Elisa Assay ◽

Expression Systems ◽

Cancer Antigen 125 ◽

Cancer Antigen ◽

Specific Proteins ◽

Size Exclusion Hplc

Abstract BACKGROUND By using proteomics and bioinformatics, we have previously identified a group of highly pancreas-specific proteins as candidate pancreatic ductal adenocarcinoma (PDAC) biomarkers. With the use of commercially available ELISAs, the performance of some of these candidates was initially evaluated in a relatively small serum cohort (n = 100 samples). This phase revealed that CUB and zona pellucida-like domains protein 1 (CUZD1) may represent a new, promising PDAC biomarker. METHODS We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay. CUZD1 was expressed in house in both bacteria and yeast expression systems. Recombinant CUZD1 and biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards, were analyzed by Western blot, size exclusion HPLC, and mass spectrometry (LC-MS Orbitrap). RESULTS We confirmed that instead of CUZD1, the commercial assay is recognizing a nonhomologous, known cancer antigen [cancer antigen 125 (CA125)]. CONCLUSIONS We conclude that poor characterization of commercial ELISA assays is a factor that could lead to false biomarker discovery. To our knowledge, this is the first report documenting that a commercial ELISA marketed for one analyte (CUZD1) may, in fact, recognize a different, nonhomologous antigen (CA125).

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Characterization of the α-mannosidase gene family in filamentous fungi: N-glycan remodelling for the development of eukaryotic expression systems

Biotechnology and Bioprocess Engineering ◽

10.1007/bf02942178 ◽

2000 ◽

Vol 5 (4) ◽

pp. 227-233 ◽

Cited By ~ 2

Author(s):

C. Joshua Eades ◽

William E. Hintz

Keyword(s):

Gene Family ◽

Filamentous Fungi ◽

Eukaryotic Expression ◽

Expression Systems ◽

Eukaryotic Expression Systems

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Characterization of Codon Usage Bias in UL13 Gene of the Duck Plague Virus

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.393-395.641 ◽

2011 ◽

Vol 393-395 ◽

pp. 641-650

Author(s):

Xi Xia Hu ◽

An Chun Cheng ◽

Ming Shu Wang

Keyword(s):

Codon Usage ◽

Codon Usage Bias ◽

Codon Position ◽

Expression System ◽

Expression Systems ◽

Yeast Expression System ◽

E Coli ◽

Synonymous Codons ◽

Suitable Expression

A comprehensive analysis of codon usage bias of DPV UL13 gene (GenBank Accession No. EU195098) was performed to provide a basis for understanding the relevant mechanism for its biased usage of synonymous codons and for selecting suitable expression systems to improve the expression of UL13 genes. Our study showed that codon usage bias of DPV UL13 gene strongly prefered to the synonymous with A and T at the third codon position. And ENC value and GC3s contents of the codon usage bias of UL13 gene in DPV were significantly different compared with those in other 21 reference herpesviruses. The phylogentic analysis about the putative protein of DPV UL13 and the 21 reference herpesviruses revealed that DPV was evolutionarily closer to the AnHV-1. In addition, the codon usage bias of DPV UL13 gene was compared with those of E. coli, yeast and human. There are 23 codons showing distinct usage differences between DPV and E. coli, 12 codons between DPV and yeast, 21 codons between DPV and human. Therefore, the yeast expression system is more appropriate for heterologous expression of the DPV UL13 gene.

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