A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.     

Comparison of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique

beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli.

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies

The expression of IgG antibodies in Escherichia coli is of increasing interest for analytical and therapeutic applications. In this work, we describe a comprehensive and systematic approach to the development of a dicistronic expression system for enhanced IgG expression in E. coli encompassing: (i) random mutagenesis and high-throughput screening for the isolation of over-expressing strains using flow cytometry and (ii) optimization of translation initiation via the screening of libraries of synonymous codons in the 5' region of the second cistron (heavy chain). The effects of different promoters and co-expression of molecular chaperones on full-length IgG production were also investigated. The optimized system resulted in reliable expression of fully assembled IgG at yields between 1 and 4 mg/L of shake flask culture for different antibodies.     

Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation.

Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.     

Investigation of the active site of Escherichia coli beta-D-galactosidase by photoaffinity labelling.

3,7-Anhydro-2-azi-1,2-dideoxy-D-glycero-L-manno-(8-3H)octitol++ + (1a) and 3-azibutyl 1-thio-beta-D-(6-3H)galactopyranoside (2a) were synthesised from the unlabelled compounds by reaction with galactose oxidase, then reduction with sodium borotritide. Whereas 1a was an efficient photoaffinity reagent for the beta-D-galactosidase from E. coli, 2a was ineffective. Three 3H-labelled peptides were isolated after digestion of the labelled enzyme with trypsin, one of which was an octapeptide (Trp 158 to Ser 165), which is remote from the segments detected as part of the active site of the enzyme.     

Characterization of an endoribonuclease, RNase N, from Escherichia coli.

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.     

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.     

Transfer and incorporation of genes controlling beta-D-galactosidase synthesis from Hfr and F' donors of Escherichia coli

Ganesan, Ann K. (Syntex Institute of Molecular Biology, Palo Alto, Calif.), and Boris Rotman. Transfer and incorporation of genes controlling beta-d-galactosidase synthesis from Hfr and F' donors of Escherichia coli. J. Bacteriol. 92:1378-1382. 1966.-Comparisons were made between Hfr(1) and F(13) donors with respect to the frequency of transfer and incorporation of genes controlling beta-d-galactosidase synthesis. The Hfr(1) donor transfers these genes as part of the chromosome, and the F(13) donor transfers them by F-duction. The criterion used for gene transfer was the acquisition by recipient cells of the ability to synthesize the enzyme, beta-d-galactosidase, measured by fluorogenic assays at the single-cell level. The criterion for incorporation was the formation of lac(+) recombinant colonies. It was found that the two types of donor showed the same frequency of gene transfer, but the probability of incorporation was 10-fold higher in F(13) matings than in Hfr(1) matings. In the former, between 46 and 97% of the merozygotes produced recombinant colonies; in the latter, 2 to 6% did so.     

A rapid immunoblotting procedure for detecting serum antibodies to the lipopolysaccharide of Escherichia coli 0157

An immunoblotting method, which can be completed in one day, is described for the detection of serum antibodies to Escherichia coli 0157 lipopolysaccharide.     

Preterm and term neonates transplacentally acquire IgG antibodies specific to LPS from Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa

High incidences of Gram-negative bacteria are found in neonatal nosocomial infections. Our aim was to investigate placental transmission of immunoglobulin G (IgG) reactive with lipopolysaccharide from Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli O111, O6 and O26. The total and lipopolysaccharide-specific IgM and IgG were determined in 11 maternal/umbilical-cord sera aged ≤33 weeks (GI); 21 aged >33 and <37 weeks (GII); and 32 term newborns (GIII). The total and lipopolysaccharide-specific IgM concentrations were equivalent in maternal sera. The total IgG concentrations were equivalent in maternal and newborn sera, with the exception of GIII newborns as compared with their mothers (P<0.0001) and with neonates from GI and GII (P<0.05). Lipopolysaccharide-specific IgG concentrations were lower in GI neonates than in their mothers (P<0.01) and lower in GII (P<0.05). Lower lipopolysaccharide-specific IgG levels were observed among neonates only for O111 in GI (P<0.05) and for O26 and Pseudomonas in GII, both as compared with GIII (P<0.05). The anti-lipopolysaccharide IgG transfer ratios were lower in GI (except for O26) and in GII (except for Klebsiella and O111) as compared with GIII (P<0.05). Our results suggest that the greater susceptibility to infections in preterm infants is influenced (besides the humoral response) by factors intrinsic and extrinsic to the condition of prematurity.     

Detection and localization of the i protein in Escherichia coli cells using antibodies.

Using antibodies raised against the purified i protein, the expression of the chromosomal uncI gene was demonstrated. The i protein was identified as a component of the cytoplasmic membrane and shown to be present in preparations of Fo or F1Fo. The protein is not associated with the F1 moiety.     

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies.

Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.     

Minimal concentration of human IgM and IgG antibodies necessary to protect mice from challenges with live O6 Escherichia coli

This work evaluated the ability of human anti-lipopolysaccharide O6 IgM and IgG antibodies to protect mice challenged with Escherichia coli serotype O6 : K2ac. Purified IgM-effluent, purified IgG, pools of normal human serum (NHS), or control group were injected into mice 18 h before challenges with O6 E. coli. Interleukin 6 and tumor necrosis factor alpha were quantified in the sera of test and control groups. All mice receiving purified IgM-effluent (66.6 mg L(-1) of anti-lipopolysaccharide O6 IgM antibodies) and NHS survived. Purified IgG (1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies) protected 87.5% of the animals. The control group showed no protective ability. The minimal concentration of anti-lipopolysaccharide O6 IgM antibodies, able to protect 50% of the animals was 33.3 mg L(-1) of purified IgM-effluent, whereas purified IgG was able to protect 50% of the animals with only 1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies. Serum from animals pretreated with purified IgM-effluent and purified IgG before challenges with lipopolysaccharide O6 did not have detectable pro-inflammatory cytokines. Hepatocytes of the control group were completely invaded by bacteria, whereas none was found in animals pretreated with purified IgM-effluent and purified IgG. Higher concentrations of anti-lipopolysaccharide O6 IgM antibodies as compared to anti-lipopolysaccharide O6 IgG antibodies were needed to protect mice from challenges with E. coli O6 serotype.     

Over-expression, purification, and characterization of aminopeptidase N from Escherichia coli

The gene from Escherichia coli encoding aminopeptidase N (PepN) was subcloned into pET-26b, and PepN was over-expressed in BL21(DE3) E. coli and purified using Q-Sepharose chromatography. This protocol yielded over 17 mg of purified, recombinant PepN per liter of growth culture under optimum conditions. Gel filtration chromatography revealed that recombinant PepN exists as a monomer. MALDI-TOF mass spectra showed that the enzyme has a molecular mass of 98,750 Da, and steady-state kinetic studies revealed that as-isolated, recombinant PepN exhibits a k(cat) of 354 +/- 11s(-1) and a K(m) of 376 +/- 39 microM when using L-alanine-p-nitroanilide as the substrate. Metal analyses demonstrated that as-isolated, recombinant PepN binds 0.5 and <0.1 equivalents of iron and zinc, respectively. The addition of Zn(II) to recombinant PepN inhibits catalytic activity, while the addition of iron causes a slight decrease or no change in activity. Further metal binding studies revealed that recombinant PepN tightly binds 5 equivalents of iron and <0.1 equivalents of Zn(II). By using this over-expression and purification system, E. coli PepN can now be obtained in quantities necessary for structural characterization and possibly inhibitor design efforts.