A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b 5 led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.Although integral membrane proteins account for almost 25% of open reading frames in fully sequenced genomes, progress on understanding their structure and function has lagged behind their soluble counterparts. In part, this is due to the difficulty in obtaining sufficient quantities of homogenous protein for in vitro studies using traditional expression systems. For example, the available space in cellular membranes, the toxic effects of competition for the membrane insertion machinery, and incorrect lipid composition for proper folding may limit the utility of Escherichia coli for eukaryotic membrane protein production [1]. Efforts to study the enzymology and structure of membrane proteins have been hindered by these difficulties over several decades.As one example, stearoyl-CoA desaturases are integral membrane proteins thought to have four trans-membrane sequences [2]. They have a conserved motif consisting of 8 His residues hypothesized to provide at least some of the ligands to a catalytically essential diiron center [2]. In 1974, Strittmatter and colleagues published a preparation of the stearoyl-CoA desaturase from the livers of starved and then fed rats [3]. This achievement ultimately permitted a number of important properties of the enzyme complex to be elucidated [4][5][6]. However, no comparable reports on the successful purification of mammalian stearoyl-CoA desaturase have arisen in the ensuing four decades.

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“…3A, also see Fig. 7 of [31] for purification of cytb5 using this approach). The electrophoresis of Fig.…”

Section: Resultsmentioning

confidence: 99%

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

Goren

Fox

2008

Protein Expression and Purification

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“…The expression plasmids used in these studies yield recombinant proteins containing an N-terminal affinity tag linked to MshB via a tobacco etch virus (TEV) protease site: pVP55A (His tag) (29), pVP56K (His-maltose-binding protein (MBP) tag) (30), and pFN18K (HaloTag, Promega). The mshB genes were amplified from genomic DNA with PmeI and SgfI restriction sites at the 5Ј-and 3Ј-ends, respectively.…”

Section: Methodsmentioning

confidence: 99%

The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

Huang

Kocabas

Hernick

2011

Journal of Biological Chemistry

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2 as their primary reducing agent and in xenobiotic metabolism for the detoxification of drugs and other toxins (1-4). MSH is likely to be critical for the survival of mycobacteria inside activated macrophages, where the mycobacteria are subjected to oxidative bursts. Consequently, the enzymes involved in MSH biosynthesis and detoxification (Fig. 1A), including the metalloenzymes N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-␣-D-glucopyranoside deacetylase (MshB) and MSH-conjugate amidase, are targets for the development of antibiotics for the treatment of diseases such as tuberculosis (5-10).The enzyme MshB catalyzes the hydrolysis of (GlcN-Ins) and acetate, the fourth overall step in MSH biosynthesis (rate-limiting step) (11). MshB is an attractive drug target because it is a metalloenzyme; there are past successes in targeting metalloenzymes, including inhibitors of carbonic anhydrase, matrix metalloproteases, and angiotensinconverting enzyme (12-15). Inhibitors of metalloenzymes typically contain a group that binds to the catalytic metal ion. Consequently, a comprehensive understanding of metalloenzyme cofactor preferences is necessary for the development of potent and specific metalloenzyme inhibitors.MshB was previously identified as a Zn 2ϩ -dependent enzyme based on the observations that the enzyme copurifies with Zn 2ϩ (Fig. 1B) and that the enzyme activity is reversibly inhibited by treatment with 1,10-phenanthroline (16 -18). On the basis of the structure of the enzyme active site, MshB is thought to catalyze the hydrolysis of GlcNAc-Ins via one of two potential chemical mechanisms using general acid-base catalysis (GABC) (19). One possible mechanism uses a single bifunctional GABC to facilitate the hydrolysis of GlcNAc-Ins, whereas the other uses a GABC pair to carry out this reaction. However, Fe 2ϩ was not examined as a potential cofactor in these experiments. Furthermore, MshB was purified using zinc immobilized metal ion affinity chromatography (IMAC) under aerobic conditions, which is biased toward zinc incorporation into metalloenzymes (16). Purified MshB contains nickel (0.82 eq) when purified using nickel IMAC (aerobic conditions) (16). There have been several examples over the last decade of Fe 2ϩ -enzymes being misidentified as exclusive Zn 2ϩ -enzymes, including peptide deformylase, S-ribosylhomocysteinase (LuxS), UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC), and possibly histone deacetylase-8 (HDAC8) (20 -27). In all these enzymes, the Fe 2ϩ cofactor is either exclusively preferred or is preferred over Zn 2ϩ under certain environmental conditions. The

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“…The digested PCR product was then ligated into the plasmids pVP55A, pVP56K, and pFN18K, which were previously treated with Sgf I and Pme I. Cloning was designed for the expression of an N-terminus tagged fusion protein. In pVP55A, the gene was expressed with an 8x-His tag, in pVP56K, the gene was expressed with an 8x-His tagged maltose binding protein (MBP), and in pFN18K, the gene was expressed with a HaloTag [19, 20]. In the vectors pVP55A and pVP56K, the inserted gene was under the control of the T5 promoter and in pFN18K, the inserted gene was under control of the T7 promoter.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of functional Leishmania major virulence factor UDP-galactopyranose mutase

Oppenheimer

Valenciano

Sobrado

2011

Biochemical and Biophysical Research Communications

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Human parasitic pathogens of the genus Leishmania are the causative agents of cutaneous, mucocutaneous, and visceral leishmaniasis. Currently, there are millions of people infected with these diseases and over 50,000 deaths occur annually. Recently, it was shown that the flavin-dependent enzyme UDP-galactopyranose mutase (UGM) is a virulence factor in Leishmania major. UGM catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose. The product, UDP-galactofuranose, is the only source of galactofuranose which is present on the cell surface of this parasite and has been implicated to be important for host-parasite interactions. The recombinant form of this enzyme was obtained in a soluble and active form. The enzyme was shown to be active only in the reduced sate. A kcat value of 5 ± 0.2s−1 and a KM value of 87 ± 11 μM were determined with UDP galactofuranose as substrate. Different from the dimeric bacterial and tetrameric fungal UGMs, this parasitic enzyme functions as a monomer.

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A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

Cited by 24 publications

References 56 publications

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

Isolation and characterization of functional Leishmania major virulence factor UDP-galactopyranose mutase

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