Heterologous gene expression in a membrane-protein-specific system.

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.     

Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state

Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.     

Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe

The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.     

An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli

Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In N. pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H. salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from H. salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in H. salinarum and E. coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.     

Bacterioopsin-mediated regulation of bacterioruberin biosynthesis in Halobacterium salinarum

Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors have important functions in all organisms. To form functional complexes, cofactor biosynthesis must be coordinated with the production of corresponding apoproteins. To examine this coordination, we study bacteriorhodopsin (BR), a light-induced proton pump in the halophilic archaeon Halobacterium salinarum. This complex consists of a retinal cofactor and bacterioopsin (BO), the BR apoprotein. To examine possible novel regulatory mechanisms linking BO and retinal biosynthesis, we deleted bop, the gene that encodes BO. bop deletion resulted in a dramatic increase of bacterioruberins, carotenoid molecules that share biosynthetic precursors with retinal. Additional studies revealed that bacterioruberins accumulate in the absence of BO regardless of the presence of retinal or BR, suggesting that BO inhibits bacterioruberin biosynthesis to increase the availability of carotenoid precursors for retinal biosynthesis. To further examine this potential regulatory mechanism, we characterized an enzyme, encoded by the lye gene, that catalyzes bacterioruberin biosynthesis. BO-mediated inhibition of bacterioruberin synthesis appears to be specific to the H. salinarum lye-encoded enzyme, as expression of a lye homolog from Haloferax volcanii, a related archaeon that synthesizes bacterioruberins but lacks opsins, resulted in bacterioruberin synthesis that was not reduced in the presence of BO. Our results provide evidence for a novel regulatory mechanism in which biosynthesis of a cofactor is promoted by apoprotein-mediated inhibition of an alternate biochemical pathway. Specifically, BO accumulation promotes retinal production by inhibiting bacterioruberin biosynthesis.     

Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His(6)). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His(6) and CFTR peptides or His(6) peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.     

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).     

Thermal motions in bacteriorhodopsin at different hydration levels studied by neutron scattering: correlation with kinetics and light-induced conformational changes.

Bacteriorhodopsin (BR) is a transmembrane protein in the purple membrane (PM) of Halobacterium salinarum. Its function as a light-driven proton pump is associated with a cycle of photointermediates which is strongly hydration-dependent. Using energy-resolved neutron scattering, we analyzed the thermal motions (in the nanosecond-to-picosecond time range) in PM at different hydration levels. Two main populations of motions were found that responded differently to water binding. Striking correlations appeared between these "fast" motions and the "slower" kinetic constants (in the millisecond time range) of relaxations and conformational changes occurring during the photocycle.     

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

---

     

The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides.

The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.     

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium The designation H. salinarium instead of the former designation H. halobium is used throughout this paper following the classification of Tindall (1992) . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.     

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.     

Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His₆). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His₆ and CFTR peptides or His₆ peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.     

Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein.

Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E. coli at 25 degrees C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP.     

Optimization of bacteriorhodopsin for bioelectronic devices

Bacteriorhodopsin (BR) is the photoactive proton pump found in the purple membrane of the salt marsh archaeon Halobacterium salinarum. Evolution has optimized this protein for high photochemical efficiency, thermal stability and cyclicity, as the organism must be able to function in a hot, stagnant and resource-limited environment. Photonic materials generated via organic chemistry have yet to surpass the native protein in terms of quantum efficiency or cyclicity. However, the native protein still lacks the overall efficiency necessary for commercial viability and virtually all successful photonic devices using bacteriorhodopsin are based on chemical or genetic variants of the native protein. We show that genetic engineering can provide significant improvement in the device capabilities of proteins and, in the case of bacteriorhodopsin, a 700-fold improvement has been realized in volumetric data storage. We conclude that semi-random mutagenesis and directed evolution will play a prominent role in future efforts in bioelectronic optimization.     

Human neutrophil response to recombinant neisserial Opa proteins

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.     

The secondary structure of bacteriorhodopsin determined by Raman and circular dichroism spectroscopy

The secondary structure of bacterio-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), has been determined by Raman spectroscopy. Additional circular dichroism (CD) measurements have revealed only negligible conformational differences between BO in apomembranes and BR in purple membranes. Therefore, the secondary structure of BR was derived from the Raman data of BO. The protein conformation was determined to consist of 72-82% helices, 2-11% beta-strands, and 11-17% beta-turns. Only about half of the helical structures correspond to alpha 1-helices, the other half possess non-alpha 1-helical structures. According to the analysis of the Raman data, the derived secondary structure of BR was obtained with high reliability for all structure classes which can be distinguished by this method within the given uncertainty range. This is a remarkable difference from recently published secondary structural data derived from CD measurements where the helix content was reported to be between 50 and 80%. The inherent experimental and methodological uncertainties of the CD-technique leading to such a range of variation are critically discussed in comparison to the method of Raman spectroscopy. The combined application of Raman and CD spectroscopy, as performed here, is demonstrated to be a substantial improvement in the secondary structure determination of retinal-containing membrane proteins. On the basis of our results, some of the recently proposed structural models of BR with a beta-strand content of more than 11% can be ruled out.     

Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.     

Structural determinants of purple membrane assembly

The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of Halobacterium salinarum. High-resolution structural studies, in conjunction with detailed knowledge of the lipid composition, make the purple membrane one of the best models for elucidating the forces that are responsible for the assembly and stability of integral membrane protein complexes. In this review, recent mutational efforts to identify the structural features of bacteriorhodopsin that determine its assembly in the purple membrane are discussed in the context of structural, calorimetric and reconstitution studies. Quantitative evidence is presented that interactions between transmembrane helices of neighboring bacteriorhodopsin molecules contribute to purple membrane assembly. However, other specific interactions, particularly between bacteriorhodopsin and lipid molecules, may provide the major driving force for assembly. Elucidating the molecular basis of protein-protein and protein-lipid interactions in the purple membrane may provide insights into the formation of integral membrane protein complexes in other systems.