Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis.

Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis.

MUP1, high affinity methionine permease, is involved in cysteine uptake by Saccharomyces cerevisiae.

Using a mutant defective in cysteine uptake, which is resistant to a toxic analog of cysteine, allylglycine, we searched for a gene that complements the defect in cysteine uptake in a yeast genomic library and found a DNA fragment causing the recovery of cysteine uptake and sensitivity to allylglycine. The gene in the fragment was identical to MUP1, the high affinity methionine permease gene. We conclude that Mup1 is a major permease in cysteine uptake.

The est1 regulation depends on the oxygen concentration in Acetobacter pasteurianus.

The regulation mechanism for expression of the ethanol inducible esterase gene, est1, was investigated in A. pasteurianus. Deletion analysis of the 5' non coding region of est1 showed that the FNR-binding consensus sequence is important in the induction of est1 by ethanol. Cells grown under oxygen starvation produced esterase-1 in not only the presence but also the absence of ethanol. These results suggest that the induction of est1-expression depends on the oxygen concentration, and the gene may be induced by a FNR-like factor activated by a decrease in the intracellular oxygen concentration.

Construction of a chimeric shuttle plasmid via a heterodimer system: secretion of an scFv protein from Bacillus brevis cells capable of inhibiting hemagglutination.

Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease. For this purpose, we attempted to produce a single chain variable fragment, scFv, which inhibited hemagglutination using the Bacillus brevis protein-producing system. To accomplish this, a novel strategy, a heterodimer system, was used for the construction of a chimeric shuttle plasmid. Initially, a set of new plasmids, kanamycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed. p15A ori was a common replication origin in these plasmids, while the pUB110 rep and minus origin (MO) were cloned into the donor plasmid. Next, the secretion domain of the B. subtilis alpha-amylase gene and the G2-4 gene, coding for the scFv protein, were cloned into the general cloning plasmid and fused by PCR. Both the donor plasmid and the general cloning plasmid containing the fused gene were digested with NotI and them ligated, a dimeric plasmid being constructed. The key restriction sites, AscI, are arranged such that the pUB110 rep-MO moiety was switched from the donor to the general cloning plasmid following AscI digestion. The chimeric shuttle plasmid was readily constructed by simple re-circularization and a B. brevis transformant producing the scFv protein in the culture fluid was isolated.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

A gene encoding phosphatidylethanolamine N-methyltransferase from Acetobacter aceti and some properties of its disruptant.

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Efficient production of N-terminally truncated biologically active human interleukin-6 by Bacillus brevis.

cDNAs encoding human interleukin 6 (hIL-6) and its variants lacking the N-terminal Pro and Pro-Val-Pro-Pro, respectively, were expressed in Bacillus brevis by using the signal peptide fusion approach. The presence of Pro at the N-terminus of the mature protein hindered the action of the Bacillus brevis signal peptidase. hIL-6 lacking the N-terminal Pro-Val-Pro-Pro was most efficiently secreted in a biologically active form and accumulated in the culture medium to a level of 200 mg per liter, which is the highest level reported for the bacterial secretion of hIL-6.

Efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression/secretion system of Bacillus brevis and its mucosal immunoadjuvanticity.

A gene encoding the mature Escherichia coli heat-labile enterotoxin B subunit (LTB) was introduced in a vector pNU212 and expressed at high levels in Bacillus brevis HPD31. The maximum amount of recombinant LTB (rLTB) secreted into the modified 5PY medium containing erythromycin was about 350 mg l(-1) when cultivated at 30 degrees C for 8 days. The rLTB purified directly from the culture supernatant by using D-galactose immobilized agarose was identical to the native LTB with respect to the molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the amino terminal amino acid sequence. Western blot analysis with antiserum to cholera toxin B subunit (CTB) indicated that rLTB had cross-reactivity to native CTB and its GM1 binding ability was almost the same as that of the CTB. The rLTB predominantly showed the pentameric form when non-boiled samples were applied to SDS-PAGE. When rLTB was administered intranasally to mice with diphtheria toxoid (D(T)), it resulted in the substantial stimulation of D(T)-specific serum IgG antibody, and in the induction of moderate levels of D(T)-specific mucosal IgA antibody responses in the nasal cavities and in the lung, suggesting that purified rLTB acts as a promising immunoadjuvant on mucosal immunizations.

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

Role of intracellular esterases in the production of esters by Acetobacter pasteurianus.

Esters are the major flavor compounds produced by Acetobacter sp. during vinegar production. The two genes encoding the esterases in the bacteria were disrupted, and the effects of the disruptions studied. When cultured in the presence of ethanol, the est1 gene-disrupted mutant (DE1K) did not produce any ethyl acetate or isoamyl acetate. However, the disruption of est2 did not affect the ester production. Ethyl acetate production by N-23 (pME122P) and DE1K (pME122P), which contain est1, was 1.7-fold higher than that by the wild type, N-23. On analyzing the relationship between ethyl acetate production and the extracellular ethanol and acetic acid concentrations, we found that the highest amount of ethyl acetate was produced when the molar ratio of ethanol and acetic acid was 1:1. These results indicate that the ester production by Acetobacter sp. is mostly catalyzed by the intracellular esterase, esterase-1, with ethanol and acetic acid used as the substrates.

A permease exhibiting a dual role for lysine and biotin uptake in Saccharomyces cerevisiae.

Among Saccharomyces cerevisiae strains each defective in one of 11 amino acid permeases, a lysine permease disruptant (DK) exhibited about 2-fold reductions in maximum cell density and fermentation ability compared to the parent in a synthetic medium. These unusual properties of DK were found to result from the requirement of biotin for growth, in contrast to the parent whose growth was not dependent on external biotin. The rate of 14C-labeled biotin uptake and the intracellular free biotin content of DK were 2-2.5 fold lower than in the parent. We suggest that lysine permease in S. cerevisiae has the ability to transport both lysine and biotin.

Human interleukin-1 receptor antagonist: large-scale expression in Bacillus brevis 47-5Q.

Interleukin-1 receptor antagonist (IL-1RA) has been used as a tool to study the biologic activity of IL-1 and as a possible therapeutic substance for inflammatory disease. To perform in vivo study, however, large quantities of IL-1RA are required. Bacillus brevis strains secrete large amounts of protein but little protease into the medium. Using B. brevis 47-5Q, we developed a large-scale expression system of human IL-1RA (HuIL-1RA). The bacteria secreted HuIL-1RA into the culture medium at very high levels, approximately 200 mg/L. The protein was isolated in one-step purification with monoclonal antibody (mAb) against HuIL-1RA. The IL-1RA molecule was determined to be functionally active by the inhibiting assay of HuIL-1-induced cell proliferation in a mouse T cell line, D10N4M.

Isolation of peptides from an enzymatic hydrolysate of food proteins and characterization of their taste properties.

Soybean protein, casein, bonito protein and chicken protein, each as foodstuff protein, were hydrolyzed with four proteinases; namely, pepsin, trypsin, alpha-chymotrypsin and bromelain. Since the chicken protein hydrolysate with bromelain possessed the most favorable umami taste, eleven peptides were isolated from the chicken protein hydrolysate by successive chromatography on ODS, Amberlite IR-120B, Amberlite IRA-410 and AG-50W; their structures were Asp-Ala, Asp-Val, Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Asp-Glu-Glu, Asp-Glu-Ser, Glu-Glu-Asn, Ser-Pro-Glu, and Glu-Pro-Ala-Asp. Many of them did not show any umami taste by themselves, but Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Asp-Glu-Glu, and Ser-Pro-Glu were recognized to enhance the umami taste of 0.02% 5'-inosine monophosphate (IMP). A combination of these peptides, especially 0.5% each of Glu-Glu, Glu-Val, Asp-Glu-Glu and Glu-Glu-Asn, with 0.02% IMP produced a delicious "full" umami taste.

Cloning and characterization of ethanol-regulated esterase genes in Acetobacter pasteurianus.

The esterase encoding genes, est1 and est2, were cloned from Acetobacter pasteurianus. Nucleotide sequence analysis of est1 revealed a gene of 954 bp, and est1 coded for an arylesterase with a molecular weight of 34863 Da consisting of 317 amino acids. The est2 gene contained an open reading frame composed of 1221 bp encoding an esterase with a molecular weight of 43389 Da consisting of 406 amino acids. The est1 gene showed some similarity, but the est2 gene showed no significant homology to other esterases reported in various microorganisms. Northern blot analysis of total RNA from A. pasteurianus revealed that transcription of the est1 gene was induced only when the cells were grown in a medium containing ethanol, and suggested that the est1 transcript is monocistronic. In contrast, transcription of the est2 gene was repressed in the presence of ethanol. In the absence of ethanol, expression of the est2-mRNA, capable of encoding a multiple number of proteins, was revealed by Northern blot analysis. In addition, deletion analysis indicated that the 5'-region of the est2 gene contained a cis-acting domain for est2 transcriptional regulation. Analysis of the est1 promoter using the chloramphenicol acetyltransferase gene as a reporter gene showed that the promoter within the 305-bp fragment upstream of the ATG initiation codon was responsible for the transcription in cells grown in the presence of ethanol. Primer extension analysis of est1-mRNA showed that the transcription initiation site was 49 bp upstream from the ATG initiation codon. The results of a gel mobility shift assay indicated that there is a regulatory protein related to est1 regulation, which may have some relation to the ethanol resistance of Acetobacter sp.

Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant.

The efficient production of a thermostable protein disulfide isomerase (PDI) was successfully achieved using the newly isolated protease-deficient mutant, Bacillus brevis 31-OK. Extracellular protease (exoprotease) activity was about a quarter of that in the parent, and the mutant was deficient in at least one of the major exoproteases. The cDNA encoding the fungal PDI was inserted downstream of the signal peptide-encoding region in an expression-secretion vector for B. brevis. Efficient production of PDI was feasible using B. brevis 31-OK as a host and modified signal sequences composed of three leucine residues inserted in the hydrophobic region of the MWP (middle wall protein) signal sequence. The maximal secretion of PDI into the culture medium was 1.1 g/l, which is about twice that by the parent strain and fifty times greater than the amount of rat and murine PDIs produced by Escherichia coli. The enzymatic properties such as the specific activity and thermal stability of the recombinant PDI are similar to those of natural PDI derived from Humicola insolens mycelia. B. brevis 31-OK was able to maintain its exoprotease activity at a low level throughout the cultivation and is considered to be useful host for production of a protease-sensitive protein and for increase of protein productivity due to stable accumulation.

Sweetness of lysozymes.

While examining the taste of various proteins, we found that hen egg-white lysozyme, a bacteriolytic enzyme, had sweetness. Lysozymes from other sources such as turkey and soft-shelled turtle also showed sweetness with different tastes, heavy or light. In contrast, human lysozyme was tasteless. The amino acid sequences of the various lysozymes were similar to that of hen lysozyme, but hen lysozyme did not show significant homology to sweet proteins.

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis.

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

Secretion of human interleukin-2 in biologically active form by Bacillus brevis directly into culture medium.

We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis.