Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

To determine the modes of three disulfide linkages in the heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6-18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138-142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6-18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6-18) are Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys.     

Mass spectrometric detection of the plasma prealbumin (transthyretin) variant associated with familial amyloidotic polyneuropathy

A plasma prealbumin variant with a methionine-for-valine substitution at position 30 is closely associated with familial amyloidotic polyneuropathy (FAP) type I. Secondary ion mass spectrometry of the tryptic digest of a carrier's prealbumin could easily detect an abnormal peptide containing the substitution besides the normal peptide. This is a sensitive and reliable method for the diagnosis of FAP.     

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.     

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3',5'-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.     

Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli

We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities. This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence. Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK. The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3. Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1). The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E. coli crude extracts. In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography. The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65. It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3. The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene. The turnover number for this conjugation reaction is 57 s-1. Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism. We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.     

Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type)

Structural studies on an amyloid fibril protein of 14 K daltons (AFj(INO] isolated from a Japanese patient who suffered from familial amyloidotic polyneuropathy were carried out to unambiguously identify its difference from normal human serum prealbumin. Sequence analyses performed by comparing peptide maps prepared from cyanogen bromide fragments and tryptic peptides of purified RCM-amyloid protein with those from RCM-prealbumin indicate that only a valine residue at position 30 in prealbumin is replaced by a methionine residue. Furthermore, it was also proved that AFj(INO) consists of four components; the prealbumin variant and its three related proteins, which are derived by successively accumulated deletion of the N-terminal three amino acid residues (Gly1, Pro2 and Thr3) from the prealbumin variant.     

Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.     

Identification and characterization of a human transthyretin variant

An apparent Mr variant of plasma transthyretin (TTR), previously detected using 2-D PAGE, is the first reported occurrence of this type of human TTR variant. We characterized the variant TTR to determine the nature of this difference. Comparative tryptic peptide maps of variant and normal TTR and sequencing of peptides which differed indicated the variant contained a single amino acid substitution of valine for tyrosine at position 116. Because such a change requires two nucleotide substitutions, we postulate the variant arose through mutation in codon 116 of a heretofore unrecognized polymorphic or rare variant allele of TTR.     

Restriction of exogenous transthyretin (prealbumin) by the endothelium of the rat choriocapillaris

The endothelium of the choriocapillaris has been shown to restrict molecules with Einstein-Stokes radii greater than or equal to 3.2 nm which correspond to minimal molecular weights of approximately 64,000-68,000 daltons. The present study was undertaken to determine if the endothelium restricts exogenous transthyretin (prealbumin), a 55,000-dalton carrier of retinol-binding protein. Rats were injected with human 125I-transthyretin which was allowed to circulate for 15 and 30 min. Chromatographic analysis demonstrated that the human transthyretin did not bind to rat blood proteins. Eye tissue from injected rats was prepared for light and ultrastructural autoradiographic analysis. Autoradiographic grains were confined to areas over the lumen of the choriocapillaris with few present on Bruch's membrane, the basement membrane common to the endothelium of the choriocapillaris and the retinal pigment epithelium. These findings demonstrate that the choriocapillaris can restrict transthyretin and suggest a possible role of its endothelium in the metabolism of retinol-carrier molecules.     

Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera-like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH₂-terminal amino acid sequence revealed homology with the toxin-coregulated pilus of Vibrio cholerae, the bundle-forming pilus of enteropathogenic E. coli and type IV pilins of some Gram-negative bacterial pathogens. The longus structural gene (IgA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K-12. The presence of IngA was restricted to human ETEC strains. In contrast to other ETEC pili, IngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immuno-prophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat-stable toxin only.     

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system

Therapeutic proteins require correct disulfide bond formation for biological activity and stability. This makes their manufacturing and storage inherently challenging since disulfide bonds can be aberrantly formed and/or undergo significant structural changes. In this paper the mechanisms of disulfide bond formation and scrambling are reviewed, with a focus on their impact on the biological activity and storage stability of recombinant proteins. After assessing the research progress in detecting disulfide bond scrambling, strategies for preventing this phenomenon are proposed.     

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.     

Characterization of shiga toxin type 2 variant B-subunit in Escherichia coli strains from asymptomatic human carriers by PCR-RFLP

Subtyping of shiga toxin type 2 variant B-subunit in 35 non-O157 and two O157 strains isolated from 37 asymptomatic human carriers yielded two strains with stx2, 10 strains with stx2c and 24 strains with stx2d genes. One isolate harboured stx2 and stx2c. The high Stx2d prevalence in asymptomatic carriers was conspicuous and may indicate a reduced pathogenicity of these toxin variants. Therefore, in order to appraise a positive STEC laboratory result, the strain must be isolated in every case. Shiga toxin types and further virulence-associated factors have to be investigated.     

Stress-governed expression and purification of human type II hexokinase in Escherichia coli

The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL21 (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK II(6xHis) existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at 18 degrees C, about 83% of HXK II(6xHis) existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at 37 degrees C. The soluble form of HXK II(6xHis) was also highly produced in the presence of 1 M sorbitol under the standard condition (37 degrees C), which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with Ni2+ ions, resulting in about 40 mg recombinant HXK II protein obtained with purity over 89% from 5 l of E. coli culture. The identity of HXK II(6xHis) was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.     

Purification of human interleukin-4 produced in Escherichia coli

An interleukin-4 (IL4)-encoding cDNA isolated from human splenocytes was used to construct an expression plasmid that directs a high-level synthesis of mature IL4 protein in Escherichia coli. The expression was under the control of the major leftward promoter, pL, of phage lambda and the phage Mu ribosome-binding site. The IL4 protein was present as insoluble inclusion bodies in the bacterial extract. The IL4 could be solubilized in 5 M MgCl2 and was purified to homogeneity by several chromatographic steps. The yield of protein from bacteria ranged between 3 and 5 mg of IL4 protein per gram of wet cells. The specific activity of the recombinant human IL4 was about the same as that of the natural product.     

Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli

In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2–10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.     

High level expression of human lipocortin (annexin) 1 in Escherichia coli

Summary Human lipocortin (annexin) 1, a member of the annexin family of phospholipid binding proteins, has previously been expressed in E. coli (Huh et al., 1990). To improve the expression level of lipocortin 1 in E. coli, several expression vectors containing either the P_L or the P_trc promoter were constructed. The highest expression level, up to 20 % of the total E. coli proteins, was obtained with plasmid pHT3. Plasmid pHT3 contains the pUC origin, the lipocortin 1 cDNA under P_trc promoter, and the lacI gene.