scholarly journals From sequences to enzymes: heterologous expression of genes from marine microbes

2021 ◽  
Author(s):  
Angelica Severino ◽  
Alessandro Coppola ◽  
Monica Correggia ◽  
Costantino Vetriani ◽  
Donato Giovannelli ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Expression System ◽  
Marine Microbes ◽  
Functional Perspective ◽  
Expression Of Genes ◽  
Protein Encoding ◽  
Protein Functions ◽  
Main Components ◽  
Encoding Genes

Heterologous expression is an easy and broadly applicable experimental approach widely used to investigate protein functions without the need to genetically manipulate the original host. The approach is used to obtain large quantities of the desired protein, which can be further analyzed from a biochemical, structural and functional perspective. The expression system consists of three main components: i) a foreign DNA sequence coding for the protein of interest; ii) a suitable expression vector; iii) a suitable host (bacterial, yeast or mammalian cells) which does not encode or express the protein of interest. Here we show how to apply an Escherichia coli-based expression system to overexpress protein encoding genes from marinemicrobes.

scholarly journals Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259837
Author(s):  
Zora Novakova ◽  
Daria Khuntsaria ◽  
Marketa Gresova ◽  
Jana Mikesova ◽  
Barbora Havlinova ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Eukaryotic Cells ◽  
Post Translational Modifications ◽  
Protoporphyrinogen Oxidase ◽  
Intracellular Targeting ◽  
Alternative Systems ◽  
Protein Functions ◽  
The Impact

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

scholarly journals TheNitrosopumilus maritimusCdvB, but Not FtsZ, Assembles into Polymers

Archaea ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Kian-Hong Ng ◽  
Vinayaka Srinivas ◽  
Ramanujam Srinivasan ◽  
Mohan Balasubramanian
Keyword(s):  
Cell Division ◽  
Heterologous Expression ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Expression System ◽  
Related Protein ◽  
Heterologous Expression System

Euryarchaeota and Crenarchaeota are two major phyla of archaea which use distinct molecular apparatuses for cell division. Euryarchaea make use of the tubulin-related protein FtsZ, while Crenarchaea, which appear to lack functional FtsZ, employ the Cdv (cell division) components to divide. Ammonia oxidizing archaeon (AOA)Nitrosopumilus maritimusbelongs to another archaeal phylum, the Thaumarchaeota, which has both FtsZ and Cdv genes in the genome. Here, we used a heterologous expression system to characterize FtsZ and Cdv proteins fromN. maritimusby investigating the ability of these proteins to form polymers. We show that one of the Cdv proteins inN. maritimus, the CdvB (Nmar_0816), is capable of forming stable polymers when expressed in fission yeast. TheN. maritimusCdvB is also capable of assembling into filaments in mammalian cells. However,N. maritimusFtsZ does not assemble into polymers in our system. The ability of CdvB, but not FtsZ, to polymerize is consistent with a recent finding showing that several Cdv proteins, but not FtsZ, localize to the mid-cell site in the dividingN. maritimus. Thus, we propose that it is Cdv proteins, rather than FtsZ, that function as the cell division apparatus inN. maritimus.

scholarly journals Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
David John Carrier ◽  
Norliza Tendot Abu Bakar ◽  
Karen Lawler ◽  
James Matthew Dorrian ◽  
Ameena Haider ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Functional Assay ◽  
Expression Systems ◽  
Heterologous Expression Systems ◽  
Membrane Spanning ◽  
High Level

Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

scholarly journals What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

2021 ◽  
Author(s):  
Blase Matthew LeBlanc ◽  
Rosamaria Yvette Moreno ◽  
Edwin Escobar ◽  
Mukesh Kumar Venkat Ramani ◽  
Jennifer S Brodbelt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphorylation State ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Protein Encoding ◽  
Small Nuclear Rnas ◽  
Rnap Ii ◽  
Carboxy Terminal ◽  
Encoding Genes

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

scholarly journals Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

2012 ◽  
Vol 79 (1) ◽  
pp. 411-414 ◽  
Author(s):  
Afonso G. Abreu ◽  
Vanessa Bueris ◽  
Tatiane M. Porangaba ◽  
Marcelo P. Sircili ◽  
Fernando Navarro-Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Markers ◽  
Protein Encoding ◽  
Encoding Genes ◽  
Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

Expression of hepatitis B viral core region in mammalian cells

1986 ◽  
Vol 6 (5) ◽  
pp. 1393-1400
Author(s):  
M J Roossinck ◽  
S Jameel ◽  
S H Loukin ◽  
A Siddiqui
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Mammalian Cells ◽  
Expression System ◽  
Core Region ◽  
Core Antigen ◽  
Northern Blot Hybridization ◽  
The Core ◽  
B Virus ◽  
Nuclease Mapping

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

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