Texte remanié de: thèse--Sc.--Paris, 1966. / C.N.R.A.O. 1172. Extrait du "Bulletin de la Société de chimie biologique" T. XLVI, 1964, pp. 867-880. Conservé sous la cote: [4° THS. Pièce. 272] et sous la cote [4° THS. Pièce. 72].

Two separate systems were developed for the expression of recombinant metalloproteins. A synthetic gene encoding the lipase-solubilized form of bovine liver microsomal cytochrome b₅ was designed and assembled for expression in E. coli. Analysis of the initial recombinant cytochrome revealed differences in several physical characteristics of the molecule compared to the authentic bovine liver species, including a reduction potential that was lower by 17 mV. Further studies showed the primary sequence of the initial recombinant differed from the authentic protein in the amidation status of three residues which, when corrected yielded a recombinant protein identical in behaviour to the authentic protein. The participation of Ser64 in the stabilization of the oxidized form of cytochrome b₅ was investigated using site-directed mutagenesis to alter this residue to Ala, which was predicted to ablate a hydrogen bond formed between the protein and heme-propionate 7. Spectroelectrochemical analysis of this variant showed that the reduction potential had been shifted downwards by 7 mV, in contrast to predictions from a structural model describing the red/ox behaviour of cytochrome b₅ (Argos and Mathews, 1975). The role of heme carboxylates in determining the reduction potential was confirmed for both the wild-type and Ala64 variants by heme replacement studies using the esterified derivative of protoporphyrin IX, suggesting that the presence of free carboxylates contributes to the stabilization of the oxidized species. In addition, constructions for the expression of the trypsin-solubilized form of bovine liver microsomal cytochrome b₅ and the erythrocytic form of human cytochrome b₅ are described. A tissue culture cell system was developed for the expression of the N-terminal half molecule of human serum transferrin. The recombinant molecule (hTF/2N) was secreted at high levels from selected eukaryotic cells, and displayed high identity with the proteolytically-derived molecule from authentic human serum transferrin as judged by sequence analysis, electrophoretic mobility and iron binding capacity. A construction for the expression of the C-terminal half molecule was assembled but failed to express recombinant protein when introduced into tissue culture cells. The production of these two heterologous expression systems allows for high-level recovery of recombinant protein and provides a convenient approach to structure-function studies employing site-directed mutagenesis techniques. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (MSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Membrane bound cytochrome b5 is a ubiquitous protein with an average molecular weight of 16 kDa. The protein is involved in a number of reactions providing electrons directly to cytochrome P450 enzymes or to other enzymes involved in lipid biosynthesis. It is also known that the protein influences the activities of certain enzymes via an allosteric effect. It has been accepted in the literature that the cytochrome b5 exists primarily in the monomeric form, however, recently it has been shown that it forms homomeric complexes in vivo. In this study, we investigate the cytochrome b5 complex formation using a variety of analytical tools. Cytochrome b5 was isolated from ovine liver microsomes and the purity verified using sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrospray ionisation mass spectrometry. The latter analysis confirmed the presence of a single heme containing protein with Mr=15865 Da, while separation on the polyacrylamide gel revealed oligomeric complex formation with the tetrameric form the most prominent oligomer. Using different and particularly harsh denaturing conditions we found that the observed oligomeric aggregates persisted, indicating highly stable complexes. The most prominent tetrameric aggregate was identified to be cytochrome b5 by mass spectrometric sequencing. Further complex formation studies, using a fluorescent dye (1-anilinonaphthalene-8-sulfonic acid) that interact with hydrophobic cavities formed during oligomerisation, provided evidence of protein assembly in oligomeric complexes or aggregation. The formation of the cytochrome b5 complexes was dependent on ionic strength and protein concentration. Previously it was shown that the hydrophobic membrane anchoring domain plays a pivotal role in the cytochrome b5’s homomeric complexes. Using a peptide (IITTIDSNSS), resembling a portion of this domain, together with circular dichroism we showed more organized structure present for the wildtype peptide vs. a mutated control peptide (LLSSLKAVAV). A modified ELISA interaction assay also revealed that the wild-type peptide had a specific interaction with cytochrome b5, providing further evidence that the membrane anchoring domain plays a role in complex formation. These studies also indicated that a hydrogen bond network in this domain may be important for the formation of the homomeric complexes of cytochrome b5. / AFRIKAANSE OPSOMMING: Membraan-gebonde sitochroom b5 is ’n alomteenwoordige proteïen met ’n gemiddelde molekulêre massa van 16 kDa. Die proteïen is betrokke in reaksies waar dit elektrone direk aan sitochroom P450 ensieme verskaf, sowel as ensieme betrokke in lipiedbiosintese. Dit is ook bekend dat die proteïen die aktiwiteite van sekere ensieme via ’n allosteriese effek beïnvloed. Dit is geredelik in die literatuur aanvaar dat sitochroom b5 as ’n monomeer voorkom, maar daar is kort gelede gerapporteer dat homomeriese komplekse in vivo vorm. In hierdie studie is die sitochroom b5-kompleksvorming ondersoek deur gebruik te maak van verskeie analietiese metodes. Sitochroom b5 is vanuit skaaplewer mikrosome geïsoleer en die suiwerheid met behulp van natrium-dodesiel-sulfaat-poliakrielamied-gel-elektroforese en elektrosproei-ionisasie massa-spektrometrie geverifieer. Met die laasgenoemde bevestig dat ’n enkele heem-bevattende proteïen met Mr =15865 teenwoordig was, terwyl poliakrielamied gel-skeiding kompleksvorming getoon het, met tetrameer as die mees prominente oligomeer. Deur verskeie denaturerings kondisies, intsluitend besondere kondisies, is gevind dat hierdie aggregate behoue bly, wat baie stabiele oligomere aandui. Die mees prominente tetrameriese aggregaat is as sitochroom b5 geïdentifiseer met behulp van massa spektrometriese volgordebepaling. Kompleksvorming is verder bewys deur ’n verdere ondersoek met behulp van ’n fluoresserende kleurstof (1-anilinonaftaleen-8-sulfoonsuur) wat met die hirdofobiese holtes, wat vorm tydens oligomermerisasie, interaksie het. Die kompleksvorming was afhanklik van ioniese sterkte, sowel as proteïenkonsentrasie. Voorheen was dit bewys dat die deurslaggewende faktor in die vorming sitochroom b5 se homomeriese komplekse die hidrofobiese membraan-anker-domein is. Deur gebruik te maak van ’n peptied (IITTIDSNSS) wat lyk soos ’n gedeelte van hierdie domein, tesame met sirkulêre dichroisme, is gewys dat meer georganiseerde struktuur teenwoordig was vir die wilde tipe peptied vs. ’n gemuteerde kontrole peptied (LLSSLKAVAV). ’n Gemodifiseerde ELISAinteraksie- essai het ook getoon dat die wilde-tipe peptied spesifieke interaksie met sitochroom b5 het, ’n verdere bewys dat hierdie membraan-anker-domein ’n rol speel in kompleksvorming. Hierdie studies het ook aangedui dat ’n waterstofbinding netwerk in die domein belangrik kan wees vir die vorming van die homomeriese komplekse van sitochroom b5.

The system of mixed function oxygenase (MFO system) participates in significant roles in the metabolism of endogenous compounds and xenobiotics. This system contains cytochrome P450, NADPH:cytochrome P450 reductase, and also there are assigned NADH: cytochrome b5 reductase and cytochrome b5. It was proved that cytochrome b5 can stimulate or inhibit cytochrome P450 (CYP)-dependent reactions and even change the ratio of resulting metabolites. The mechanism of cytochrome b5 action has not been fully elucidated yet. Elucidation of protein-protein interactions in MFO system and determination of topology of this system could explain the mechanism of cyt b5 action. The covalent cross- linking technique is suitable method for identifying protein-protein interactions within the membrane. Cytochrome b5 contains 3 methionines and in 2 cases the methionines are localized in a short hydrophobic C-terminal membrane anchor. Interactions with cytochrome P450 in the membrane environment can be identified by substitution of two methionine for photoactivatable analogue of methionine (photo-methionine) and subsequent photoactivation. This work is focused on expression and isolation of photo-cytochrome b5 (photo- cyt b5), cytochrome b5 analogue with incorporated photo-methionine. Conditions for photo-methionine...

Les besoins en protéines recombinantes dans les diverses activités des bio-industries a considérablement augmenté ces dernières années. Cependant, les procédés de leur production sont encore limités par le manque de marqueurs permettant de suivre l'expression et la purification des protéines d'intérêt, la formation des corps d'inclusion et les faibles degrés de pureté.Afin de pallier à ces difficultés, nous avons développé et mis en œuvre un nouveau procédé de production et de purification de protéines recombinantes chez Escherichia coli. Ce procédé est basé sur l'utilisation d'une cassette d'expression appelée Multitags et sur le clivage par une TEV protéase immobilisée sur une matrice de streptavidine. Le Multitags comporte, à partir de son extrémité N-terminale, un double tag d'affinité (10xHis et SBP), le domaine de fixation de l'hème du cytochrome b5 et le site de clivage de la TEV protéase. En utilisant deux modèles différents de protéine d'intérêt (MyRIP et la Pfu DNA polymérase), nous avons montré l'efficacité du cytochrome b5 dans le suivi visuel et quantitatif par mesure d'absorbance des différentes étapes de production et de purification. Nous avons obtenu plus de 90% de chacune des deux protéines de fusion dans la phase soluble. L'application d'une chromatographie double via les deux tags d'affinité 10xHis et SBP a permis d'atteindre un degré de pureté du Multitags-MyRIP et du Multitag-Pfu de 99%. Nous avons construit des colonnes protéolytiques en produisant la TEV protéase sauvage et sa version mutée (S219V) en fusion avec le Streptag II et en immobilisant ces enzymes par affinité sur colonne de streptavidine-agarose. La caractérisation des colonnes protéolytiques et leur application aux protéines recombinantes d'intérêt modèles ont montré l'avantage de cette méthode d'immobilisation en termes d'activité protéase retenue, de stabilité des enzymes, de leur réutilisation et de simplification du schéma de purification et de récupération des protéines d'intérêt à haut degré de pureté. En conclusion, ces travaux de thèse ont permis de développer et de valider des outils innovants pour l'expression et la purification de protéines recombinantes. / In recent years, the need for recombinant proteins has substantially increased in various bio-industry activities. However, actual recombinant processes are still limited by the lack of markers allowing real-time expression and purification monitoring of target proteins, by inclusion bodies formation and by low quality of purity of the products. To overcome these difficulties, we have developed a new process for production and purification of recombinant proteins in Escherichia coli. The method combines the use of a multifunctional expression cassette, termed Multitags and an immobilized modified TEV protease on a streptavidin matrix. The Multitags contains, its N-terminus, two affinity purification tags (10xHis and SBP) and as a marker tag, the heme-binding domain of cytochrome b5 followed by the TEV cleavage site. Using two model proteins (MyRIP and Pfu DNA polymerase), we have demonstrated the visual and the quantitative monitoring capability of the cytochrome b5 during the expression and purification steps. When expressed in E. coli KRX more than 90% of both fusion proteins were produced in a soluble form. In addition, high purity (99%) of Multitags-MyRIP and Multitags-Pfu was achieved after two consecutive affinity purification steps using the dual affinity tag. We also produced the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence and realized their affinity immobilization on a streptavidin-agarose matrix. The characterization of the proteolytic columns and their application to the recombinant model proteins demonstrated the advantage of this immobilization method in terms of retaining activities, enzyme stabilities, possibility of reuse and simplification of the cleavage downstream steps.In conclusion, this study allowed the development and the validation of innovative tools for expression and purification of recombinant proteins.

Cytochromes P-450 (P450s) belong to haemoprotein superfamily and they are responsible for metabolism of a wide variety of compounds, among others many drugs and carcinogens. P450s serve as the terminal oxidases in the mixed function oxidase system in cooperation with a redox partner NADPH: cytochrome P450 reductase (CPR) providing input of two electrons to the reaction cycle of P450. The CPR can be substituted by other redox partner of P450, cytochrome b5 (cyt b5), to deliver the second electron. Three dimensional structure of P450 is required in order to fully understand its reaction mechanism. At the present time, a homology model of cytochrome P-450 2B4 (CYP 2B4) is available in our laboratory. In this study, the mapping of interaction domain between CYP 2B4 and cyt b5 employing a crosslinking agent EDC to form amide bonds between close complementary charged amino acid side chains was the first goal. We have identified five interacting amino acid pairs in total using mass spectrometry (MS). The second research interest was to verify and refine the CYP 2B4 model using a photoaffinity labelling with N-(p-azidobenzyl)-N-methyl-p-aminophenylamine probe. This photoreactive probe is known as CYP 2B4 ligand binding to the central iron atom of haem. After photoactivation the arginine 197 was found by MS...

Mixed-function oxygenase systém (MFO systém) plays a vital role in the metabolism of a variety of both endogenous substrates and xenobiotics. This membrane systém consists of cytochrome P450s, NADPH:cytochrome P450 oxidoreductase (POR), cytochrome b5 and NADH:cytochrome b5 oxidoreductase (b5R). Cytochrome P450 catalyzes a monooxygenation of a substrate, while POR and cytochrome b5 represent its redox partners. Cytochrome b5, itself having a redox partner in b5R, effects the reactions catalyzed by the MFO system in various ways, through mechanisms that are not fully understood. This paper focuses on the purification of b5R and POR from rabbit liver. The microsomal fraction obtained by differential centrifugation contained 42 mg of protein per ml. From a portion of the microsomal fraction, b5R was obtained using chromatography on DEAE-Sepharose, CM-Sepharose and 5'-ADP agarose columns. The yield was 0,3 % of ferricynide-reductase activity and the product contained several contaminants in the molecular weight range of 50-70 kDa. A second purification of b5R from the microsomal fraction was carried out using a column of DEAE-Sepharose directly connected to a 5'-ADP agarose column. The b5R product was purified with a yield of 10,9 % and it once again contained several contaminants in the molecular...

Cytochrome b5 is a small heme protein. There are three isoforms present in human organism, one is located in the membrane of endoplasmic reticulum (microsomal cyt b5), second in the outer mitochondrial membrane and the third (soluble form) was found in cytoplasm of matured erythrocytes. The main role of cytochrome b5 is to transport single electron in various reactions including cytochrome P450-dependent reactions. First aim of the thesis was to prepare and to isolate the soluble form of rabbit microsomal cytochrome b5, using heterologous expression in Escherichia coli strain BL-21 Gold. The plasmid pET22b containing synthetic gene for rabbit microsomal cytochrome b5, lacking the sequence encoding the membrane associated C-terminal domain, was used as an expression vector. The second aim was to synthesize the expression vectors carrying genes for human microsomal and erythrocytic cytochromes b5. These genes were prepared by gene synthesis, ligated to cloning vector pUC19, amplified in E. coli DH5α competent cells and their sequences were verified by DNA sequencing. Consequently the pET22b expression vectors containing genes for human microsomal and erythrocytic cytochrome b5 were constructed and finally their suitability for heterologous expression was evaluated. Keywords: Heterologous expression,...

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal