1.1 Heterologous Expression of Genes in Mammalian Cells

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.

Download Full-text

Heterologous expression of genes from the fumonisin degradation gene cluster of Sphingomonas spp. MTA144 and activity of the catabolic enzymes

New Biotechnology ◽

10.1016/j.nbt.2009.06.444 ◽

2009 ◽

Vol 25 ◽

pp. S132-S133 ◽

Cited By ~ 1

Author(s):

D. Hartinger ◽

S. Heinl ◽

R. Grabherr ◽

G. Schatzmayr ◽

W.D. Moll

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Catabolic Enzymes ◽

Expression Of Genes

Download Full-text

Heterologous expression of microbial flavohemoglobin can modulate the effects of nitric oxide in mammalian cells

The FASEB Journal ◽

10.1096/fasebj.24.1_supplement.871.2 ◽

2010 ◽

Vol 24 (S1) ◽

Author(s):

Christine Elissa Eyler ◽

Michael T. Forrester ◽

Anita B. Hjelmeland ◽

Jeremy N. Rich

Keyword(s):

Nitric Oxide ◽

Heterologous Expression ◽

Mammalian Cells

Download Full-text

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

PLoS ONE ◽

10.1371/journal.pone.0259837 ◽

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.

Download Full-text

Oxygen sensing and molecular adaptation to hypoxia

Physiological Reviews ◽

10.1152/physrev.1996.76.3.839 ◽

1996 ◽

Vol 76 (3) ◽

pp. 839-885 ◽

Cited By ~ 815

Author(s):

H. F. Bunn ◽

R. O. Poyton

Keyword(s):

Transcription Factors ◽

Mammalian Cells ◽

Critical Role ◽

Hypoxia Inducible Factor ◽

Signal Transduction Pathway ◽

Adaptation To Hypoxia ◽

Adaptive Responses ◽

Expression Of Genes ◽

Activation Of Transcription ◽

Activation Of Oxygen

This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

Download Full-text

Interactions of GMP with Human Glrx3 and with Saccharomyces cerevisiae Grx3 and Grx4 Converge in the Regulation of the Gcn2 Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.00221-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Mónica A. Mechoud ◽

Nuria Pujol-Carrion ◽

Sandra Montella-Manuel ◽

Maria Angeles de la Torre-Ruiz

Keyword(s):

Saccharomyces Cerevisiae ◽

Heterologous Expression ◽

Mammalian Cells ◽

Iron Homeostasis ◽

Life Extension ◽

Iron Sulfur Clusters ◽

Content Type ◽

Functional Conservation ◽

Iron Sulfur ◽

Cell Life

ABSTRACT The human monothiol glutaredoxin Glrx3 (PICOT) is ubiquitously distributed in cytoplasm and nuclei in mammalian cells. Its overexpression has been associated with the development of several types of tumors, whereas its deficiency might cause retardation in embryogenesis. Its exact biological role has not been well resolved, although a function as a chaperone distributing iron/sulfur clusters is currently accepted. Yeast humanization and the use of a mouse library have allowed us to find a new partner for PICOT: the human GMP synthase (hGMPs). Both proteins carry out collaborative functions regarding the downregulation of the Saccharomyces cerevisiae Gcn2 pathway under conditions of nutritional stress. Glrx3/hGMPs interact through conserved residues that bridge iron/sulfur clusters and glutathione. This mechanism is also conserved in budding yeast, whose proteins Grx3/Grx4, along with GUA1 (S. cerevisiae GMPs), also downregulate the integrated stress response (ISR) pathway. The heterologous expression of Glrx3/hGMPs efficiently complements Grx3/Grx4. Moreover, the heterologous expression of Glrx3 efficiently complements the novel participation in chronological life span that has been characterized for both Grx3 and Grx4. Our results underscore that the Glrx3/Grx3/Grx4 family presents an evolutionary and functional conservation in signaling events that is partly related to GMP function and contributes to cell life extension. IMPORTANCE Saccharomyces cerevisiae is an optimal eukaryotic microbial model to study biological processes in higher organisms despite the divergence in evolution. The molecular function of yeast glutaredoxins Grx3 and Grx4 is enormously interesting, since both proteins are required to maintain correct iron homeostasis and an efficient response to oxidative stress. The human orthologous Glrx3 (PICOT) is involved in a number of human diseases, including cancer. Our research expanded its utility to human cells. Yeast has allowed the characterization of GMP synthase as a new interacting partner for Glrx3 and also for yeast Grx3 and Grx4, the complex monothiol glutaredoxins/GMPs that participate in the downregulation of the activity of the Gcn2 stress pathway. This mechanism is conserved in yeast and humans. Here, we also show that this family of glutaredoxins, Grx3/Grx4/Glrx3, also has a function related to life extension.

Download Full-text

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

RSC Advances ◽

10.1039/c9ra06908a ◽

2019 ◽

Vol 9 (60) ◽

pp. 35124-35134 ◽

Cited By ~ 1

Author(s):

Yun-Ming Qiao ◽

Rui-Lin Yu ◽

Ping Zhu

Keyword(s):

Agrobacterium Tumefaciens ◽

Secondary Metabolites ◽

Heterologous Expression ◽

Gene Targeting ◽

Fungal Genome ◽

P Gene ◽

Expression Of Genes ◽

Foreign Dna ◽

Fungal Secondary Metabolites

Gene targeting involves integration of foreign DNA into the fungal genome by several strategies including Agrobacterium tumefaciens-mediated transformation (ATMT).

Download Full-text

TheNitrosopumilus maritimusCdvB, but Not FtsZ, Assembles into Polymers

Archaea ◽

10.1155/2013/104147 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 6

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.

Download Full-text