Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure- function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system

Mass spectrometry-based proteomics in the life sciences: a review

Proteomics concerns itself with the characterization and function of all cellular proteins, the ultimate determinants of cellular function. Mass spectrometry has emerged as the preferred method for in-depth characterization of the protein components of biological systems. Using mass spectrometry, key insights into the composition, regulation and function of molecular complexes and pathways have been gained. Now days, mass spectrometry-based proteomics has become an indispensable tool in the cellular and molecular life sciences. This review discusses current mass spectrometry-based proteomics technologies

EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin

Electron paramagnetic resonance (EPR) data reveal large differences between the ferric (C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo-and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins

Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family

<p>Abstract</p> <p>Background</p> <p>The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. <it>In silico </it>analysis of the genome of <it>C. elegans </it>identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in <it>C. elegans</it>.</p> <p>Results</p> <p>All 33 <it>C. elegans </it>putative globin genes are transcribed. The translated sequences have the essential signatures of single domain <it>bona fide </it>globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of <it>C. elegans</it>. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in <it>daf-2(e1370)</it>/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates <it>hif-1 </it>transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of <it>C. elegans</it>, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect.</p> <p>Conclusion</p> <p>We found that all 33 putative globins are expressed, albeit at low or very low levels, perhaps indicating cell-specific expression. They show wide diversity in gene structure and amino acid sequence, suggesting a long evolutionary history. Ten globins are responsive to oxygen deprivation in an interacting HIF-1 and DAF-16 dependent manner. Globin ZK637.13 is not responsive to oxygen deprivation and regulated by the Ins/IGF pathway only suggesting that this globin may contribute to the life maintenance program.</p

Poperinge, Sint-Janscollege (W.-Vl.): een eeuwenlange pottenbakkerijsite. Eindverslag van een toevalsvondst oude regelgeving

De uitbreiding van het gebouwenbestand van het Sint-Janscollege aan het Burgemeester Bertenplein in Poperinge noopte in 2015 gedurende een maand tot een uitgebreid toevalsvondstonderzoek. Dit terrein bevond zich op het achtererf van pottenbakkerij Dupont die tot in 1975 actief bleef. De vroegste vermelding van de potterie dateert uit 1543. De onderzochte zone, de achterzijde van het perceel, kreeg pas in de loop van de 19de eeuw permanente bewoning. De oudste aangetroffen muren zijn wellicht de restanten van de kleiwerkplaats die hier rond 1850 werd opgericht. Vóór 1850 was dit een open zone vol kuilen en depressies waar afval van de pottenbakkerijsite werd gestort. De vele kuilen, depressies en lagen leverden een enorme hoeveelheid vondstmateriaal op, vooral aardewerk. Het betreft zowel huishoudelijk gebruiksaardewerk, bouwkeramiek als productiematerieel. De bestudeerde contexten vertegenwoordigen drie tijdsblokken: 14de vroege 15de eeuw, tweede helft 15de vroege 16de eeuw, en 18de eeuw. De gedetailleerde aardewerkstudie leverde niet alleen inzichten op over deze Poperingse pottenbakkerijsite, haar evolutie en de pottenbakkersactiviteiten zelf. Minstens even belangrijk is de bijdrage over de evolutie van de aardewerkproductie in de regio, over verschillende periodes van de 14de eeuw tot in de 18de eeuw heen

A phylogenomic profile of globins

BACKGROUND: Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. RESULTS: A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. CONCLUSION: Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals

The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification

<p>Abstract</p> <p>Background</p> <p>Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite <it>Mermis nigrescens</it>. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. <it>In silico </it>analysis of the genome of <it>Caenorhabditis elegans </it>revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles.</p> <p>Results</p> <p>In the present study we have analyzed whole genomic data from <it>C. briggsae</it>, <it>C. remanei</it>, <it>Pristionchus pacificus </it>and <it>Brugia malayi </it>and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the <it>C. elegans </it>globin complement, with even distantly related nematodes harboring orthologs to many <it>Caenorhabditis </it>globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the <it>Caenorhabditis </it>lineage. We also show that the <it>Caenorhabditis </it>globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection.</p> <p>Conclusion</p> <p>Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to <it>Caenorhabditis</it>. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.</p

CO rebinding kinetics and molecular dynamics simulations highlight dynamic regulation of internal cavities in human cytoglobin

Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein

A Hemoglobin with an Optical Function

Hemoglobins are best known as oxygen transport pro-teins. Here we describe a hemoglobin from the parasitic nematode Mermis nigrescens (Mn-GLB-E) that has an optical, light shadowing function. The protein accumu-lates to high concentration as intracellular crystals in the ocellus of mature phototactic adult females while also being expressed at low concentration in other tis-sues. It differs in sequence and expression pattern from Mn-GLB-B, a second Mermis globin. It retains the struc-ture and oxygen-binding and light-absorbing properties typical of nematode hemoglobins. As such, recruitment to a shadowing role in the eye appears to have occurred by changes in expression without modification of bio-chemistry. Both globins are coded by genes interrupted by two introns at the conserved positions B12.2 and G7.0, which is in agreement with the 3exon/2intron pat-tern model of globin gene evolution