Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes

Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes. Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase. This protein, LacZ:HP, synthesized in E. coli and purified from a crude E. coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5. Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein. LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production. These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.     

Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids

Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauchi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536- 19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from cDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli beta-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in ER targeting.     

Cytochrome b5 and a recombinant protein containing the cytochrome b5 hydrophobic domain spontaneously associate with the plasma membranes of cells

Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10(-9) to 8.10(-9) cm2 s-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

A discrete stage of baculovirus GP64-mediated membrane fusion

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.     

The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins.

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.     

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.     

Membrane topology of Escherichia coli diacylglycerol kinase.

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.     

ATP synthase from bovine mitochondria. The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs

ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only. Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme. Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species. Partial protein sequences have been determined by direct methods. On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs. The sequences of the two proteins have been deduced from their DNA sequences. Subunit b is 214 amino acid residues in length and has a free N terminus. Subunit d is 160 amino acid residues long. Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides. The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic. This N-terminal hydrophobic region may be folded into an alpha-helical structure spanning the lipid bilayer. In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts. The b subunit in E. coli forms an important structural link between the extramembrane sector of the enzyme F1, and the intrinsic membrane domain, FO. It is proposed that the bovine mitochondrial subunit b serves a similar function. If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E. coli enzyme. Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.     

The membrane-interactive tail of cytochrome b(5) can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum

Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b(5) indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins. Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences. A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b(5), including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b(5) in the endoplasmic reticulum membrane. Precytochrome b(5) was cotranslationally translocated across the endoplasmic reticulum membrane. The signal-processed cytochrome b(5) was integrally anchored in the membrane with the globular domain facing the lumen. Thus, the topology of the signal sequence-directed cytochrome b(5) in the microsomal vesicle was reversed with respect to that of the native form. Posttranslational incubation of the precytochrome b(5) with microsomes resulted in a "loose" incorporation of the unprocessed form onto the surface of the vesicle. Our findings suggest that the membrane-insertion sequence of cytochrome b(5) has a functional stop-transfer sequence. We discuss the implications of these findings with respect to selective targeting of cytochrome b(5) to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.     

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11beta-hydroxysteroid dehydrogenase, isozyme 1 (11beta-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11beta-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy and electron microscopic immunogold labeling. The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl group and N-terminal 30-residue membrane anchor. The orientation of the cytochrome b5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.     

Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis

We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b(5) (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b(5) conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a "hydrophobic tag" for recruitment to the liposome surface.     

The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme

Rat liver alpha-mannosidase II, a hydrolase involved in the processing of asparagine-linked oligosaccharides, is an integral membrane glycoprotein facing the lumen of Golgi membranes. We have previously shown (Moremen, K. W., and Touster, O. (1986) J. Biol. Chem. 261, 10945-10951) that mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes will result in the cleavage of the intact 124,000-dalton alpha-mannosidase II subunit, releasing a 110,000-dalton hydrophilic polypeptide which contains the catalytic site. Consistent with the removal of a membrane binding domain, the chymotrypsin-generated 110,000-dalton peptide was found exclusively in the aqueous phase in Triton X-114 phase separation studies, whereas the intact enzyme was found in the detergent phase. Taking advantage of this conversion in phase partitioning behavior, a purification procedure was developed to isolate the 110,000-dalton proteolytic digestion product as a homogeneous polypeptide for further characterization and protein sequencing at a yield of greater than 65% from a rat liver Golgi-enriched membrane fraction. An improved purification procedure for the intact enzyme was also developed. The two forms of the enzyme were compared yielding the following results. (a) The catalytic activity of the intact and cleaved forms of alpha-mannosidase II were indistinguishable in Km, Vmax, inhibition by the alkaloid, swainsonine, and in their activity toward the natural substrate GlcNAc-Man5GlcNAc. (b) Both the intact and cleaved forms of the enzyme appear to be disulfide-linked dimers. (c) The two forms of the enzyme contain different NH2-terminal sequences suggesting that the cleaved NH2 terminus contains the membrane-spanning domain. (d) Additional peptide sequences were obtained from proteolytic fragments and cyanogen bromide digestion products in order to create a partial protein sequence map of the enzyme. These results are consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored to the lumenal face of Golgi membranes through an NH2-terminal hydrophobic membrane-anchoring domain.     

Genetic fusions of globular proteins to the epsilon subunit of the Escherichia coli ATP synthase: Implications for in vivo rotational catalysis and epsilon subunit function

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme. According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to epsilon generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.     

The stability and functional properties of proteoliposomes mixed with dextran derivatives bearing hydrophobic anchor groups.

Liposomes composed of Escherichia coli phospholipid were coated with polysaccharides bearing hydrophobic palmitoyl anchors. The effect on the stability of liposomes without or with integral membrane proteins was investigated. A high concentration of hydrophobized dextrans protected the liposomes against detergent degradation, decreased the fluidity of the membranes, prevented fusion of the liposomes and enhanced their stability. Proteoliposomes containing beef heart cytochrome-c oxidase and the lactose transport carrier of E. coli were similarly affected by coating with the dextrans. Under these conditions both membrane proteins were still active. Long-term stability of the coated liposomes was obtained only in the absence of the integral membrane proteins.     

Expression of human erythrocyte NADH-cytochrome b5 reductase as an alpha-thrombin-cleavable fused protein in Escherichia coli

Recombinant fused protein containing human erythrocyte NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2.) was produced in Escherichia coli, which was linked to the NH2 terminus of beta-galactosidase of the vector pUC13 via a recognition sequence of alpha-thrombin. Cleavage of purified fused protein with alpha-thrombin yielded the enzyme whose apparent molecular weight (32,000) was the same as the native enzyme. The amino-acid sequence from Phe-1 to Leu-10 was determined to be identical to that of the authentic enzyme. The purified enzyme showed an identical absorption spectrum and similar catalytic properties to the native enzyme. Establishment of the expression system would make it possible to determine the reaction mechanism of the enzyme.