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Functional expression and characterization of a bacterial light-harvesting membrane protein in escherichia coli and cell-free synthesis systems

Biosci. Biotechnol. Biochem., 68 (9), 1942–1948, 2004 Functional Expression and Characterization of a Bacterial Light-harvestingMembrane Protein in Escherichia coli and Cell-free Synthesis Systems Yuichiro SHIMADA, Zheng-Yu WANG,y Yushi MOCHIZUKI,Masayuki KOBAYASHI, and Tsunenori NOZAWA Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University, Sendai 980-8579, Japan Received May 13, 2004; Accepted June 22, 2004 Heterologous expression of a bacterial light-harvest- bacteria can be classified into two major types: a core ing (LH) integral membrane protein was attempted complex (LH1) located in close proximity to the RC, using Escherichia coli cells and cell-free synthesis and a peripheral complex (LH2) associated with the core systems prepared from E. coli extracts. The -apopro- complex.1,2) Both LH1 and LH2 are oligomers made up tein of LH1 complex from purple photosynthetic of a structural subunit consisting of a pair of highly bacterium Rhodospirillum rubrum was overexpressed hydrophobic membrane apoproteins, called and , as a recombinant protein with a histidine (His6) tag with molecular masses of 5 to 7 kDa together with added to the carboxyl terminus. Both of the expression pigment molecules, bacteriochlorophyll (BChl) a, and systems produced -apoprotein in a fully functional carotenoids. In bacterial LH complexes, both the and form as can judged by its ability to form a structural apoproteins are shown to contain a single membrane- subunit with native -apoprotein and the pigment spanning helix as the major structural element.3,4) The molecule bacteriochlorophyll a. The expression product LH1 complex can be reconstituted in vitro from its in E. coli appears to be located in the inner cell separated individual components.5,6) This provides a membrane and can be almost completely extracted by convenient way of studying pigment-protein interaction 0.5% (w/v) Triton X-100. Circular dichroism measure- through the use of various modified proteins and ment indicated that the expressed -apoproteins from pigment molecules.7,8) So far, preparation of the bacte- both systems had -helical contents essentially identical rial LH membrane proteins has relied largely on with that of the native one. About two thirds of the - homologous expression using photosynthetic bacterial apoprotein expressed in E. coli was found to have the cells. The advantage of this method is that functionally amino terminal methionine residue modified by a active protein complexes can be obtained in a pigment- formyl group. About one third of the -apoprotein associated form, and their properties, along with their expressed in the cell-free system was found to be interaction with other complexes, can be investigated in oxidized at the side chain of the amino terminal the native environment. But, the purification procedure methionine residue. Functional expression of the - is usually cumbersome for obtaining large amounts of apoprotein using the cell-free system provides an useful highly purified samples. More seriously, introduction of example for producing highly hydrophobic integral mutations into the LH protein often results in drastic membrane proteins with relatively large quantities reduction of the expression yield. As an alternative, sufficient for biophysical and structural analysis.
chemical synthesis has been used to produce differentportions of the LH protein in order to evaluate the relative contributions of various structural elements.9,10) plex; heterologous expression; cell-free syn- This method is powerful for producing short polypep- tides, but carries a risk of forming misfolded structuresfor the larger membrane proteins.
The light-harvesting (LH) antenna serves as a mo- In this study, we describe the results of other options lecular apparatus for collecting light energy and trans- for producing large quantities of bacterial LH1 mem- ferring it to the photosynthetic reaction center (RC) for brane proteins using Escherichia coli and cell-free charge separation and subsequent cyclic electron trans- expression systems. E. coli has been proven to be the port. The LH complexes from purple photosynthetic most efficient and successful host for the large-scale y To whom correspondence should be addressed. Fax: +81-22-217-7278; E-mail: wang@biophys.che.tohoku.ac.jpAbbreviations: BChl, bacteriochlorophyll; CD, circular dichroism; GuHCl, guanidine hydrochloride; HPLC, high-performance liquid chromatography; IPTG, isopropyl- -D-thiogalactopyranoside; LH, light-harvesting; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; OG, n-Octyl -D-glucopyranoside; PCR, polymerase chain reaction; RC, reaction center Expression and Characterization of LH1 Apoprotein production of water-soluble proteins from diverse enhanced E. coli extracts for coupled in vitro tran- sources. But it has been used much less extensively as scription/translation reactions. The native apoprotein a host for expressing integral membrane proteins, and was isolated and purified as described previously.20) production of large amounts of active membraneproteins by overexpression is still far from routine.11) Construction of His-tagged LH1 Expression Plas- The main reason for this appears to be the limited mid. Because the mature apoprotein was identified by capacity of E. coli to incorporate membrane proteins amino acid sequencing to contain 52 residues,21) i.e., 10 into its cytoplasmic membrane since most E. coli residues from the C-terminus corresponding to the gene cytoplasmic membrane proteins, unlike periplasmic sequence were apparently removed by C-terminal and outer membrane proteins, are non-abundant.12) An processing, we focused on the expression of the attempt to express the and apoproteins of a bacterial apoprotein corresponding to its mature form. A fragment LH1 complex in E. coli was made by co-expressing the of 165 bp was amplified by polymerase chain reaction pufBA gene in combination with polyclonal antibody (PCR) from chromosomal DNA of R. rubrum using the detection.13) The amount of the proteins expressed was following primer pairs: 50-CATATGTGGCGCATTT- estimated to be about 0.1% of total soluble protein, but GGCAGC-30 (primer 1) and 50-CTCGAGCGAGGTCT- no characterization was carried out on the expressed GGACGG GCTTC-30 (primer 2) according to the LH1 apoproteins. For larger membrane proteins, ex- published sequence of the pufA gene.22) The primers pression in E. coli tends to give inclusion bodies, or no corresponding to the 50 ends were designed to introduce over-expression is achieved. Refolding and reconstitu- a NdeI site in primer 1 and a XhoI site in primer 2 tion of the E. coli-produced membrane protein inclusion respectively. The PCR fragment was digested with bodies were demonstrated for a 25-kDa light-harvesting NdeI/XhoI and inserted into the multiple cloning site of complex II (LHC2) apoprotein from plant chloroplast expression vector pET-20b(þ) (Novagen Inc., Madison, using nickel chelating chromatography.14) On the other Wisconsin, U.S.A), which carries a hexa-histidine (His6) hand, although cell-free expression of integral mem- sequence at the C-terminus. The resulting construct brane proteins has been employed extensively to study encodes a fusion protein composed of the 52 amino acid protein insertion and assembly,15–17) large-scale cell-free apoprotein followed by one leucine, one glutamine, expression is still difficult for preparing samples in quantities sufficient for biophysical measurement. Com-parable yields were reported for bacteriorhodopsin (bR) Overexpression and Purification of the His6-Tagged obtained from in vitro and in vivo expression systems, LH1 Apoprotein. For expression in E. coli, the and the bR expressed in cell-free systems was examined plasmid constructed was transformed into E. coli host using FTIR spectroscopy.18) Recently, the cell-free strain BL21(DE3)pLysS. The culture was grown at synthesis system has been shown to be capable of 37 C in the presence of ampicillin (100 g/ml) and producing milligram quantities of soluble proteins,19) but chloramphenicol (34 g/ml) until A600 reached 0.8.
it has not been investigated whether similar results can Expression of the protein was induced by adding be obtained for integral membrane proteins using this isopropyl- -D-thiogalactopyranoside (IPTG) at a final system. In this study, we choose the apoprotein concentration of 1 mM and the culture was further (6.1 kDa) of the LH1 complex from purple photo- incubated for 7 h. The cells were harvested by centri- synthetic bacterium Rhodospirillum rubrum as a model fugation (10,000 g) at 4 C for 10 min, and the pellet was for heterologous expression using both the E. coli and suspended in sonication buffer (50 mM Tris, 150 mM cell-free systems. We attempted to compare the struc- NaCl, pH8.0). The cell suspension was sonicated on ice tural and functional properties of the products expressed and then centrifuged at 14,4000 g, 4 C for 1 h. The by addressing the following aspects: (1) expression precipitate was resuspended in solubilization buffer yields, (2) location of the expressed proteins, (3) (20 mM Tris, 500 mM NaCl, pH7.5) containing 0:5$ conformation and ability to form functional complexes 2:0% (w/v) Triton X-100. Solubilization was carried out and (4) influence of the histidine-tag at the C-terminus.
at 22 C for 1 h followed by centrifugation at 14,4000 g,4 C for 1 h. The supernatant was loaded on a 1ml Ni- chelated IDA agarose resin column (His.Bind Resin,Novagen, Madison, Wisconsin, U.S.A) equilibrated with Materials. All chemicals used were obtained from the solubilization buffer. The column was washed with Sigma Chemical Co. (U.S.A) and Wako Pure Chemical 20 mM Tris buffer (pH7.5) containing 500 mM NaCl, Industries, Ltd. (Japan), unless otherwise noted. Re- 50 mM imidazole, and 0.5% (w/v) Triton X-100. The striction and ligation enzymes were purchased from His6-tagged apoprotein was eluted with elution buffer TaKaRa Bio Inc. (Osaka, Japan) and Roche (Basel, (20 mM Tris, pH7.5, 500 mM NaCl, 200 mM imidazole, Switzerland). For cell-free expression, Rapid Trans- 0.5% (w/v) Triton X-100). The eluted protein was lation System (RTS) E. coli high-yield kits (HY 100 and dialyzed three times against deionized water at 4 C in a 500) and the RTS 500 control unit (Roche Diagnostics, dialysis membrane tube (Mw cut-off 3500, Spectrum Mannheim, Germany) were used. The kits included Laboratories, Compton, California, U.S.A), each time for 3 h. The purified protein was precipitated by adding of the expressed protein with native apoprotein and an equivolume of cold acetone and placed at À30 C for BChl a followed the same procedure as described 3 h. The samples were lyophilized to dryness and stored at À30 C. For cell-free expression using the RTSE. coli HY500 kit, the synthesis reaction was initiated by mixing the E. coli lysate, amino acid mix, and energycomponents with the constructed plasmid in a 1 ml The pET-20b(þ) vector is known as a powerful reaction compartment. Substrates and energy compo- system for the cloning and expression of recombinant nents were supplied by a way of continuous-exchange proteins in E. coli with high expression levels and via a semipermeable membrane, and at the same time precise control of induction. But the high expression potentially inhibitory reaction by-products were diluted levels tend to result in the formation of inclusion bodies.
via diffusion through the same membrane into a 10 ml Hence we first investigated the localization of the LH1 feeding compartment. The reaction was conducted at protein expressed in E. coli cells by employing either 30 C for 24 h at a stirrer speed of 150 rpm. The reaction 6 M guanidine hydrochloride (GuHCl) or 0:5$2:0% mixture was centrifuged at 144,000 g at 4 C for 1 h.
(w/v) Triton X-100 for solubilization of the expressed Precipitate was solubilized in the solubilization buffer at products. The results are shown in Fig. 1. After 22 C for 1 h followed by centrifugation at 144,000 g, disruption of the cells followed by centrifugation at 4 C for 1 h. The expressed protein contained in the 144,000 g for 1 h, expressed apoprotein was found in supernatant was purified with a Ni-IDA agarose column the precipitate. Triton X-100 was shown to be efficient in a way similar to that of the protein expressed in for solubilizing the protein, and 0.5% (w/v) was sufficient for extracting all of the expressed product. Incontrast, 6 M GuHCl failed to solubilize the apoprotein Characterization of the expressed LH1 apoprotein.
expressed in E. coli. This result indicates that the A reverse-phase HPLC column (Source 5RPC ST4.6/ apoprotein was expressed in the cell membrane rather 150, Amersham Pharmacia Biotech, Uppsala, Sweden) than in the form of inclusion bodies. After His-tag was used to investigate the elution behavior of the affinity chromatography, the expressed protein was expressed protein at a flow rate of 0.7 ml/min and a purified to a single band, as was confirmed by column temperature at 20 C. The solvent and solvent Coomassie-stained SDS-PAGE (Fig. 1, middle). For gradient used were described previously.23) Expression the cell-free synthesis system, overexpression of the yields were evaluated by BCA protein assay reagent protein was also observed. In a small-scale purification (Pierce, Rockford, Illinois, U.S.A). Mass measurements experiment, most of the expressed product was found in were performed using MALDI-TOF/MS (REFLEX III, the insoluble fraction after low-speed centrifugation of Bruker Analytic, Rheinstetten, Germany) by the method the reaction mixture at 14,000 g for 10 min, but a small described previously.23) Circular dichroism (CD) spectra fraction remained in the supernatant (Fig. 1, right).
were recorded on a J-720w spectropolarimeter (JASCO, Thereafter, the reaction mixture was centrifuged at Tokyo Japan) with a scan speed of 5 nm/min, band 144,000 g, 4 C for 1 h, and the His-tagged protein was width of 1 nm and resolution of 0.5 nm. Reconstitution purified by the same procedure as that for the protein Identification of the Expressed Products.
Left: Western blot analysis of the gene products expressed in E. coli on 20% SDS polyacrylamide gel. The membrane fraction was solubilized by 6 M GuHCl and Triton X-100 at different concentrations. The letters P and S represent precipitate and soluble fractions respectively, after thesolubilization treatment. Middle: Coomasie brilliant blue stained 20% SDS-PAGE of the purified -apoprotein expressed in E. coli withmolecular markers. Right: Western blot analysis of the gene product expressed in the cell-free synthesis system on 20% SDS polyacrylamide gel.
Lane 1 and lane 2 represent soluble and insoluble fractions respectively, after centrifugation at 14,000 g at 4 C for 10 min.
Expression and Characterization of LH1 Apoprotein expressed in E. coli cells. The expression yields were both systems had much shorter elution times compared calculated from all detergent-solubilized proteins and to that of native protein. Two fractions were observed were determined to be 1.2 mg/(l medium) and 0.7 mg/ for the expressed products from both expression sys- (ml lysate) for the E. coli and cell-free systems tems. To clarify their identities, these fractions were collected and subjected to MALDI-TOF/MS measure- The expressed proteins were examined by reverse- ment. Figure 3 shows the TOF/MS results. The F1 phase HPLC chromatography. The results are shown in fraction from the E. coli expression system had a Fig. 2 together with the chromatogram of the native molecular mass of 7142 Da, consistent with the calcu- LH1 apoproteins. Due to the hydrophilic nature of the lated value of an unmodified LH1 protein based on the His6-tag at the C-terminus, the apoprotein expressed in gene sequence (7141.2 Da, including the N-terminalMet). The molecular mass of the F2 fraction was 28 Dagreater than that of F1, suggesting that the F2 fractionfrom the E. coli expression system was a formylatedform of the F1 fraction. The slightly longer HPLCelution time for a formylated species was also observedfor the LH1 apoprotein from the thermophilic sulfurbacterium Thermochromatium tepidum.25) The majorfraction (F2) from the cell-free expression system had amolecular mass of 7174 Da. Because the proteincontains no cysteine residue in its sequence and due tothe experimental errors, this component is thought tocorrespond to the protein with an N-terminal formy-lated Met residue, although the measured mass wasslightly larger than the expected value (7170 Da). TheF1 fraction had a molecular mass 16 Da greater than thatof F2, indicating that F1 was an oxidized species of F2.
This result is in agreement with a previous study23)showing that the N-terminal methionine oxidation of the protein resulted in a slightly shorter elution time thanthat observed in this study.
Since the apoprotein expressed in E. coli appears to Reverse-phase HPLC Chromatograms of the LH1 -Apopro- be inserted into the cell membrane, we need to know teins Expressed in E. coli (a) and the Cell-free Synthesis System (b), whether it has a properly folded structure and the ability Along with That of the Native Apoproteins of LH1 Complex (c).
to form functional complexes with apoprotein and A linear gradient from 60% to 90% of organic solvents containing acetonitrile/2-propanol (2:1) and 0.1% trifluoroacetic acid was used pigment molecules. CD measurement was used to quantify the secondary structure of the expressed MALDI-TOF/MS Spectra of the Individual Fractions of the LH1 -Apoproteins Expressed in E. coli (a) and the Cell-free Synthesis Absorption Spectra of the Reconstituted B820 Subunits at CD Spectra of the LH1 -Apoproteins Expressed in E. coli Room Temperature Using the LH1 -Apoproteins Expressed in (thick solid line) and the Cell-free Synthesis System (dotted line), E. coli (solid line) and the Cell-free Synthesis System (dashed line) together with That of the Native -Apoprotein (thin solid line).
with the Native -Apoprotein and BChl a in 0.9% (w/v) OG All samples were dissolved and measured in a mixed organic solvent containing acetonitrile/2-propanol (2:1) and 0.1% trifluoro-acetic acid.
C-terminal His-tag had no marked effect on function-ality.
products. Figure 4 shows the CD spectra of the ex-pressed proteins, together with that of the native protein for comparison. The CD spectral shapes of the proteins expressed in the E. coli and cell-free systems Overexpression of integral membrane proteins to were characterized by an -helical structure, and were obtain milligram quantities for biochemical and bio- essentially identical to that of the native protein. The physical studies is usually a difficult task. Even when -helix contents were calculated to be about 43%$49% large amounts of protein are available, the hydrophobic for the expressed proteins. These results suggest that nature of membrane proteins tends to result in formation the secondary structure is well retained in the expressed of improper folding or inclusion bodies, and the problem membrane proteins. The protein is known to form a is further complicated by the selection of detergents for structural subunit (B820) in vitro with the protein and solubilization because irreversible denaturation may BChl a, which is characterized by an absorption occur during the isolation and purification processes of maximum at 820 nm.5,7–10,24,26) The intrinsic size and the membrane proteins. Several photosynthesis-related molecular weight of the B820 subunit were recently membrane proteins and the bacteriorhodopsin overex- determined unambiguously by small-angle neutron pressed in E. coli and cell-free systems were found to scattering, indicating that the subunit consists of one form inclusion bodies, and subsequent refolding was pair of proteins and two BChl a molecules.20) required to restore their functionality.14,18) We found in Figure 5 shows the absorption spectra of the reconsti- this study that the apoprotein was expressed in both tuted B820 complexes using the His-tagged apopro- the E. coli and cell-free systems in relatively large teins from both E. coli and cell-free systems with the amounts sufficient for biophysical and structural analy- native apoprotein of R. rubrum and BChl a in 0.9% sis. More importantly, the His-tagged products were (w/v) n-octyl -D-glucopyranoside (OG) solutions.
expressed in the active state and can be purified easily.
Homogeneous spectral shapes were observed for these The apoprotein expressed in E. coli appeared to be reconstituted complexes with Qy absorption maxima at localized in the host membrane, since the 6 M GuHCl 820 nm. Because it is known that the Qy electron solution failed to solubilize the expressed product. This transition is highly sensitive to the coordination state result was in agreement with a previous observation13) between the pigment molecule (BChl a) and the that the LH1 apoproteins of R. rubrum coexpressed membrane apoproteins7–10,24) and the absorption spec- in E. coli are located in the inner membrane and cannot trum has been widely used as the major means to be removed by washing with high salt of high concen- examine the function-structure relationship of the light- trations. Another study on the import and assembly of harvesting subunit complex,24,26,27) the results of this the LH proteins in the membrane systems16) also showed study indicate that the structural elements responsible that a 6 M solution of urea did not destroy membrane for pigment binding and interaction with the protein integrity and cannot extract the incorporated, integral were maintained in the expressed proteins and that the LH1 proteins. We found that the apoprotein can Expression and Characterization of LH1 Apoprotein efficiently be solubilized by 0.5% (w/v) Triton X-100, a widely used detergent for extracting integral membraneproteins. This detergent was used throughout the sub- Zuber, H., and Cogdell, R. J., Structure and organization sequent purification experiments. No deleterious effects of purple bacterial antenna complexes. In ‘‘AnoxygenicPhotosynthetic Bacteria’’, eds. Blankenship, R. E., were observed for the expressed products.
Madigan, M. T., and Bauer, C. D., Kluwer Academic It was somewhat surprising that the LH1 apopro- Publishers, Dordrecht, Netherlands, pp. 315–348 (1995).
teins, though extremely hydrophobic, were expressed in Robert, B., Cogdell, R. J., and von Grondelle, R., The a fully functional form in both the E. coli and cell-free light-harvesting system of purple bacteria. In ‘‘Light- systems. From the CD spectra, essentially the same helix Harvesting Antennas in Photosynthesis’’, eds. Green, B.
contents were obtained for the expressed and native R., and Parson, W. W., Kluwer Academic Publishers, proteins (Fig. 4). It was found that the expressed Dordrecht, Netherlands, pp. 169–194 (2003).
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shortened inside the core region within the N-terminal Parkes-Loach, P. S., Sprinkle, J. R., and Loach, P. A., domain. This behavior was explained in terms of the loss Reconstitution of the B873 light-harvesting complex ofRhodospirillum rubrum from the separately isolated - of potential ion-pairing and/or hydrogen-bonding inter- and -polypeptides and bacteriochlorophyll a. Biochem- actions between the and proteins.26) These results are consistent with those of this study. The C-terminal His- Davis, C. M., Parkes-Loach, P. S., Cook, C. K., tags in the expressed proteins were found to have no Meadows, K. A., Bandilla, M., Scheer, H., and Loach, marked influence on the subunit formation. In an in vivo P. A., Comparison of the structural requirements for expression experiment28) on a RC-LH1 related mem- bacteriochlorophyll binding in the core light-harvesting brane protein, PufX, responsible for efficient exchange complexes of Rhodospirillum rubrum and Rhodobacter of ubiquinone/ubiquinol molecules between the QB site sphaeroides using reconstitution methodology with bacteriochlorophyll analogs. Biochemistry, 35, 3072– as judged by its ability to complement the growth defect Todd, J. B., Parks-Loach, P. A., Leykam, J. F., andLoach, P. A., In vitro reconstitution of the core and of a Rhodobacter sphaeroides strain lacking the pufX peripheral light-harvesting complexes of Rhodospirillum gene. This provides a convenient tool not only for high molischianum from separately isolated components.
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