Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

The gene encoding dihydrolipoyl transacetylase from Azotobacter vinelandii. Expression in Escherichia coli and activation and isolation of the protein

The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase.

The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector. The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme. The enzyme produced in E. coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates. Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates. When activity was expressed in E. coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.     

Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein.

A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.     

The priB gene encoding the primosomal replication n protein of Escherichia coli.

The gene encoding protein n of the Escherichia coli primosome has been discovered in the rpsF-rpsR-rplI ribosomal protein operon and designated priB. The low copy number of PriB protein and the distinctive codon usage of its gene argue against its being a ribosomal protein. A strain which overproduces PriB was constructed and has been used to purify the protein to homogeneity. The overproduced protein behaves like that purified from wild-type cells.     

Expression of a synthetic gene encoding human transthyretin in Escherichia coli

Transthyretin is an amyloidogenic protein that causes human amyloid polyneuropathy and senile systemic amyloidosis as a result of the deposition of normal and/or mutant transthyretin in the form of amyloid fibrils. A high-expression plasmid of human transthyretin was constructed in order to facilitate the study of amyloid fibril formation of this protein. The transthyretin gene was constructed by an assembly of eight chemically synthesized oligonucleotides and amplified by polymerase chain reaction, and the amplified gene was inserted into an Escherichia coli expression vector. The expression plasmid was transformed into M15 cells and the gene product was expressed as a polyhistidine-tagged fusion protein. Purified recombinant transthyretin was obtained by one-step nickel chelation affinity chromatography and the production level of the protein was 130mg per 1L of culture. Furthermore, the expressed protein showed the same characteristics in terms of tetramer formation at neutral pH and amyloid formation at acidic pH as did the authentic human transthyretin. This system will enable biophysical and structural studies of this protein to be advanced.     

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.     

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli has been achieved. Although the protein is inserted into the bacterial cytoplasmic membrane, formation of a functional Fo complex was not observed.     

Expression of the transglutaminase gene in Escherichia coli.

1. The cDNA gene coding for the enzyme transglutiminase (EC 2.3.2.13) was cloned into the pUC18 oriented for expression from the lac promoter. 2. DNA sequencing of the 5' end showed that the cDNA was missing the sequence coding of the N-terminal 30 amino acids. 3. The truncated gene was then cloned into pKK233-2, and the recombinant product was produced in Escherichia coli. 4. A gene construct coding for the complete protein was generated by inserting an oligonucleotide for the missing 30 amino acids into the Eco RI site of the pUC18 clone. 5. A consensus Shine-Dalgarno sequence and translational start codon were positioned at the 5' end of the linker. 6. Immunoblotting experiments of E. coli JM105(pUC18-TGase) indicated the expression of the transglutaminase gene. 7. The cell lysate as well as the partially purified transglutaminase showed no detectable enzyme activity.     

Expression of a gene for glucan-binding protein from Streptococcus mutans in Escherichia coli

The structural gene for a glucan-binding protein (GBP) of Streptococcus mutans has been inserted into a bacteriophage lambda vector and expressed in Escherichia coli K12. Lysates of E. coli infected with the recombinant phage contain an antigenic protein of the same size as S. mutans GBP. The GBP synthesized in E. coli can be affinity-purified on immobilized glucan and antiserum raised against it has been shown to precipitate fructosyltransferase activity from S. mutans.     

Purification and properties of an oxygen-evolving Photosystem II reaction-center complex from spinach

A chlorophyll-protein complex, capable of photochemical water oxidation and consisting of only one extrinsic protein of 33 kDa in addition to six intrinsic proteins of the Photosystem II reaction center, has been isolated from spinach thylakoids by digitonin extraction, performed at pH 6.0, followed by chromatographic separations using DEAE-Toyopearl 650S as described briefly (Tang, X.S. and Satoh, K. (1985) FEBS Lett. 179, 60–64). The protein complex contained approx. 3–4 manganese atoms, 2 mol plastoquinone-9 and 2 mol low-potential forms of cytochrome b-559 heme per mol of the photoactive primary acceptor, Q_A. The oxygen evolution of the complex was highly stimulated by the presence of CaCl₂ and stabilized by glycerol; the typical rate of 400–500 μmol O₂ per mg Chl per h was attained with 2,5-dichlorobenzoquinone and potassium ferricyanide as electron acceptors in the presence of 50 mM CaCl₂. The protein complex exhibited a dark-stable EPR Signal II; the microwave power saturation profile of the signal was almost identical with that of oxygen-evolving membrane preparations. The multiline EPR signal ascribable to Kok's S₂-state was elicited in this protein complex by illumination at 200 K, as in membrane preparations. These results indicate that the basic machinery of photosynthetic water oxidation is preserved in an almost intact state in the isolated chlorophyll-protein complex.     

Extrinsic 33-kilodalton protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction

The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.