Correctly Processed Research Articles

Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence

The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6) – three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30 – obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6.The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments

A Novel Approach for Secretion of Heterologous Proteins with Correct N-Terminal Processing by Using α-Factor Pre Sequence in Pichia pastoris

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ~4.7 mg L⁻¹. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.

Long-term Delivery of Nerve Growth Factor by Encapsulated Cell Biodelivery in the Göttingen Minipig Basal Forebrain

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

Mammalian alpha-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical beta-bulge are totally conserved in the otherwise diverse sequences of all known mammalian alpha-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg(6)-Glu(14) salt bridge in human alpha-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, whereas significantly reducing the oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg(13)-Gln(14) peptide bond in the loop region, a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against Staphylococcus aureus but substantially enhanced the killing of Escherichia coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature alpha-defensin in vivo.

The Role of Amino-Terminal and Carboxy-Terminal Extensions in the Processing and Translocation of a Plant Proteinase (Actinidin) Expressed in the Yeast Saccharomyces cerevisiae

A plant proteinase gene naturally occuring in the Kiwi fruit plant (Actinidia chinensis) has been expressed in a yeast Saccharomyces cerevisiae. Different gene constructions consisting of different portions of the whole actinidin-encoding gene have been created and expressed using an expression-secretion yeast vector. It was observed that the amino- and carboxy-terminal extensions of the actinidin-encoding gene were required for the correct expression of the gene in yeast. A gene construction lacking both amino- and C-terminal extensions did not result in a detectable protein product. Similarly, a gene construction consisting of the amino-terminal extension plus mature actinidin-encoding DNA did not result in a detectable expression. However, intracellular expression was observed when a gene construction consisting of mature actinidin-encoding DNA plus C-terminal extension portion was employed. The expressed polypeptide was found however not to be correctly processed as it had a bigger size than the native actinidin. The correctly processed polypeptide was expressed intracellularly when the full-length actinidin cDNA was expressed in a vacuolar protease-proficient yeast strain. However, when a vacuolar protease-deficient yeast strain was employed, it was found that the precursor protein was not correctly processed, suggesting that the actinidin precursor had entered the vacuole and undergone proteolytic processing. The full-length actinidin cDNA consisted of the amino-terminal extension DNA, mature actinidin-encoding DNA, and C-terminal extension DNA. The results thus suggested that both amino- and C-terminal extensions were required for correct expression and processing of actinidin in yeast. The intracellular expression also suggested that the actinidin-encoding sequences contain intracellular targeting sequences which override the secretion signal included in the expression-secretion vector.

The Soluble Sema Domain of the RON Receptor Inhibits Macrophage-stimulating Protein-induced Receptor Activation

RON is a receptor tyrosine kinase of the MET family that is involved in cell proliferation, cell survival, and cell motility in both normal and disease states. Macrophage-stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. RON is a trans-membrane heterodimer comprised of one alpha- and one beta-chain originating from a single-chain precursor and held together by several disulfide bonds. The intracellular part of RON contains the kinase domain and regulatory elements. The extracellular region is characterized by the presence of a sema domain (a stretch of approximately 500 amino acids with several highly conserved cysteine residues), a PSI (plexin, semaphorins, integrins) domain, and four immunoglobulin-like folds. Here we show that a soluble, secreted molecule representing the sema domain of RON (referred to as ron-sema) has a dominant negative effect on the ligand-induced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. Results suggest that the sema domain of RON participates in ligand binding by the full-length receptor. The ability of ron-sema to suppress growth of MSP-responsive cells in culture, including cancer cells, points to a potential therapeutic use of this molecule, and forced expression of it could potentially be used as a gene therapy tool for treating MSP-dependent types of cancer.

Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity

In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S1 mapping we defined the 3′-end at 8423(±1); thus CI RNA is 79(±1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5′-end and the CCA sequence at the 3′-end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.

Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide.

Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.

Processing, targeting, and antifungal activity of stinging nettle agglutinin in transgenic tobacco.

The gene encoding the precursor to stinging nettle (Urtica dioica L. ) isolectin I was introduced into tobacco (Nicotiana tabacum). In transgenic plants this precursor was processed to mature-sized lectin. The mature isolectin is deposited intracellularly, most likely in the vacuoles. A gene construct lacking the C-terminal 25 amino acids was also introduced in tobacco to study the role of the C terminus in subcellular trafficking. In tobacco plants that expressed this construct, the mutant precursor was correctly processed and the mature isolectin was targeted to the intercellular space. These results indicate the presence of a C-terminal signal for intracellular retention of stinging nettle lectin and most likely for sorting of the lectin to the vacuoles. In addition, correct processing of this lectin did not depend on vacuolar deposition. Isolectin I purified from tobacco displayed identical biological activities as isolectin I isolated from stinging nettle. In vitro antifungal assays on germinated spores of the fungi Botrytis cinerea, Trichoderma viride, and Colletotrichum lindemuthianum revealed that growth inhibition by stinging nettle isolectin I occurs at a specific phase of fungal growth and is temporal, suggesting that the fungi had an adaptation mechanism.

Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris.

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buffered minimal medium using either the alpha-factor preprosequence with and without the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native signal peptide. Whereas ASP2 secreted using the alpha-factor preprosequence with the spacer peptide showed N-terminal heterogeneity, the recombinant protein using the two other secretion peptides was correctly processed. Mass spectrometry showed that the protein secreted using the natural peptide sequence had a mass of 13,695.1 Da, in perfect agreement with the measured molecular mass of the native protein. These data showed a native-like processing and the three disulfide bridges formation confirmed by sulfhydryl titration analysis. After dialysis, the recombinant protein was purified by one-step anion-exchange chromatography in a highly pure form. The final expression yield after 7-day fermentation was approximately 150 mg/liter. To our knowledge, this is the first report of the use of a natural insect leader sequence for secretion with correct processing in P. pastoris. The overproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein.

A novel method for increasing production of mature proteins in the periplasm of Escherichia coli.

A novel strategy to obtain high-level production of mature proteins exported to the periplasm of Escherichia coli is described. It is based on a modified signal sequence generated by insertion of a coding sequence of the polypeptide precursor of interest at the BamHI site of the commercial vector pQE-30 resulting in an addition of a dodeca-peptide (MRGSH6GS) at the N-terminus of the precursor. The modification does not affect correct processing of the modified signal nor proper folding of the target protein, resulting in an untagged native product. The method is simple for avoiding onerous optimization of translation initiation and screening of host stains. The usefulness of this method is illustrated by overexpression of DsbC and DsbA. Induced by 0.01 mM IPTG at 37 degrees C, proteins were overproduced to comprise 20-30% of the total cellular proteins, and more than 95% of the expressed proteins were correctly processed and exported into the periplasm with yields of more than 100 mg per liter culture.

Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human beta2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris.

Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae alpha-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the beta2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the alpha-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the beta2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of beta2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems. Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence. Here, the Kex2 endopeptidase failed to process the alpha-factor leader correctly. Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing. In contrast, the beta2-adrenergic receptor fusion to the alpha-factor leader was correctly processed by the internal Kex2 endopeptidase. The Kex2-processed beta2-adrenergic receptor was not glycosylated. In conclusion, the high-level production of the two receptors in P. pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.

Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import, processing, assembly and stability

The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.

Translocation and processing of various human parathyroid hormone peptides in Escherichia coli are differentially affected by protein-A-signal-sequence mutations.

Two staphylococcal protein-A signal sequences were constructed and tested for function in Escherichia coli, after being linked to human parathyroid hormone (hPTH) cDNAS representing the intact form (1-84 amino acids) and two N-terminal (1-37 and 1-7 amino acids) peptides. One signal sequence was identical to the wild type, and the other signal contained a deletion of 12 bp at the 3' end. The truncated hPTH cDNAs were fused at their 3' ends to IgG-binding domains (ZZ) derived from protein A in order to facilitate purification and characterization. The expression plasmid pSPTH, containing the wild-type signal sequence, secreted efficiently the intact recombinant hPTH (1-84) into the medium. Plasmids containing the truncated hPTH genes after the wild-type signal, gave rise to hPTH-ZZ hybrid proteins which were correctly processed at the N-terminal, but the major fractions appeared in the periplasmic compartment. In contrast, the plasmid pS'PTH which harboured the 4-amino-acid signal deletion did not promote a uniform secretion of intact hPTH (1-84) to the medium, but released a non-processed form both into the periplasmic compartment and to the medium. The related plasmids pS'PTH37ZZ and pS'PTH7ZZ with the mutated signal sequence gave rise to small or trace amounts of unprocessed forms of fusion proteins in the medium and periplasm, thus the secretion competence was markedly reduced. Thus, for correct N-terminal processing, we conclude that the amino acid sequence in the signal adjacent to the expressed protein, is a key determinant. However, release into the medium or periplasmic space appeared to be dependent also on protein folding, irrespective of signal-sequence cleavage. Furthermore, we observed that the peptides with the wild-type signal sequence and correct N-terminal processing, were the only forms that showed internal cleavage of hPTH. Uncleaved signals may contribute to folding characteristics of the ensuing protein and e.g., prevent internal proteolysis.

A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1–37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1–37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.

Expression and Processing of an Arabidopsis 2S Albumin in Transgenic Tobacco

2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleavages. In order to determine if this process is correctly carried out in transgenic plants, the gene AT2S1 encoding an Arabidopsis thaliana 2S albumin isoform has been expressed in transgenic tobacco. Initial experiments using a reporter gene demonstrated that the AT2S1 promoter directs seed specific expression in both transgenic tobacco and Brassica napus plants. The entire AT2S1 gene was then transferred into tobacco plants, where it showed a tissue specific and developmentally regulated expression. Arabidopsis 2S albumin accumulates up to 0.1% of the total high-salt extractable seed protein. Protein sequencing demonstrated that the amino termini of the two Arabidopsis 2S albumin subunits were correctly processed, suggesting that the protease(s) necessary for posttranslational processing of 2S albumin precursors may display common specificities among different dicot plant species. Immunocytochemical studies showed that the Arabidopsis 2S albumin is localized in the protein body matrix of tobacco endosperm and embryo. Correct processing and targeting of the 2S albumin in transgenic plants suggests that modified versions could be expressed, allowing the study of 2S albumin processing and in particular the possible roles of the processed fragments in protein stability and/or targeting.

Mitochondrial import of rat pre-ornithine transcarbamylase: accurate processing of the precursor form is not required for uptake into mitochondria, nor assembly into catalytically active enzyme.

Mitochondrial uptake of the cytoplasmically synthesized precursor of the mammalian enzyme ornithine transcarbamylase is mediated by an N-terminal leader sequence of 32 amino acids. In the mitochondrial matrix, the precursor form is processed to the mature subunit by proteolytic removal of this pre-sequence and in the enzyme from rat liver it has been suggested that this occurs in a two-step process which involves an intermediate cleavage at residue 24. We show that deletion of residues 20-26 spanning this intermediate cleavage site prevents correct processing to the mature subunit but it does not prevent mitochondrial targeting and internalization or assembly of the incorrectly processed product into a catalytically active enzyme. The incorrectly processed enzyme, which is larger than the normal mature enzyme, is nevertheless more susceptible to proteolytic degradation in permanently transfected human cells than the correctly processed enzyme.

Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (bamase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the bamase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of bamase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for bamase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of bamase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the bamase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence ( phoA). Cells containing this construct secreted correctly processed bamase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/1. Bamase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active bamase (His-102). The cloning and expression of bamase in E. coli will allow detailed analysis of bamase protein folding by molecular genetic approaches.

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.

Accurate processing and pseudouridylation of chloroplast transfer RNA in a chloroplast transcription system

The trancription of a cloned trnV1-trnN1-trnR1 cluster from Euglena gracilis chloroplast (ct) DNA and the processing of a tRNA(Val)-tRNA(Asn)-tRNA(Arg) polycistronic precursor were studied in a spinach ct transcription extract. A soluble ct RNA polymerase selectively transcribes the trnV1-trnN1-trnR1-trnL1 locus in the EcoG fragment from the Euglena ct genome. Restriction enzyme modified templates and RNA fingerprint analysis were used to confirm that the tRNA genes were correctly transcribed. The tRNA(Val)-tRNA(Asn)-tRNA(Arg) polycistronic precursor transcribed by RNA polymerase III in a HeLa cell extract was used as a substrate to demonstrate that a ct tRNA precursor molecule is correctly processed by the ct tRNA processing enzymes. The oligonucleotide pattern of tRNAs processed in vitro from the tRNA(Val)-tRNA(Asn)-RNA(Arg) polycistronic precursor is indistinguishable from tRNA(Val), tRNA(Asn) and tRNA(Arg) transcribed by the ct RNA polymerase and processed in the ct transcription extract. The 3'-CCAOH is added to the tRNAs by a 3' nucleotidyltransferase after correct processing of the 3' terminus. Correct pseudouridylation was demonstrated for uridine residues in a tRNA(Met) m molecule transcribed from a spinach ct trnM1 locus. Thus, the enzymatic activities involved in tRNA biosynthesis in vitro include DNA-dependent (tDNA) RNA polymerase, a 5'-processing activity (RNase P-like), a 3'-exonuclease, an endoribonuclease involved in 3'-tRNA maturation, a tRNA nucleotidyltransferase, and pseudouridylate synthetase.