Periplasmic Extracts Research Articles

The cytochrome complex SoxXA of Paracoccus pantotrophus is produced in Escherichia coli and functional in the reconstituted sulfur-oxidizing enzyme system

The heterodimeric c-type cytochrome complex SoxXA of Paracoccus pantotrophus was produced in Escherichia coli. The soxX and soxA genes, separated by two genes in the sox gene cluster of P. pantotrophus, were fused with ribosome binding sites optimal for E. coli and combined to give soxXA in pRD133.27 . The cytochrome complex SoxXA was produced in E. coli M15 containing pRD133.27, pREP4 encoding the Lac repressor and plasmid pEC86, carrying essential cytochrome c maturation genes. SoxX and SoxA were formed in a ratio of about 2.5:1. SoxA appeared to be unstable when not complexed with SoxX. The cytochrome complex SoxXA, purified to homogeneity from periplasmic extracts of E. coli M15 (pRD133.27, pREP4, pEC86), exhibited identical biochemical and biophysical properties as compared to SoxXA of P. pantotrophus. Moreover, this cytochrome complex was shown to be equally catalytically active with respect to rates and reactivity with different sulfur substrates in the reconstituted sulfur-oxidizing enzyme system using homogeneous Sox-proteins of P. pantotrophus. Homogeneous SoxX was catalytically inactive.

A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F ab fragments and leads to improved recombinant immunoglobulin stability

Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F v (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F ab fragments. pASK85 bears coding sequences for murine constant domains including a His 6 tag at the carboxyl-terminal end of the constant heavy chain domain. The anti- s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F ab fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F ab fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 μg/l×OD 550 purified F ab fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F ab was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F ab fragment exhibited significantly improved long-term stability.

Cytochrome complex essential for photosynthetic oxidation of both thiosulfate and sulfide in Rhodovulum sulfidophilum.

Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation. A thiosulfate-induced cytochrome c has been purified from the photosynthetic alpha-proteobacterium Rhodovulum sulfidophilum. This cytochrome c(551) is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit. The cytochrome c(551) structural genes are part of an 11-gene sox locus. Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination. A soxA mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide. Cytochrome c(551) is thus essential for the metabolism of both these sulfur species. Periplasmic extracts of wild-type R. sulfidophilum exhibit thiosulfate:cytochrome c oxidoreductase activity. However, such activity can only be measured for a soxA mutant strain if the periplasmic extract is supplemented with purified cytochrome c(551). Gene clusters similar to the R. sulfidophilum sox locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate–polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.

The Early Interaction of the Outer Membrane Protein PhoE with the Periplasmic Chaperone Skp Occurs at the Cytoplasmic Membrane

Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm. The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region. In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE. The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.

Processing of DNase Domain during Translocation of Colicin E7 across the Membrane of Escherichia coli

Translocation of colicin across the membrane of sensitive cells has been studied extensively. However, processing of the toxicity domain of colicin during translocation has been the subject of much controversy. To investigate the final translocation product of colicin across the membrane of Escherichia coli, an endogenously expressed His-tagged Im7 protein was constructed to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E. coli cells. As a result, a final processed DNase domain of ColE7 was identified in the intracellular space of the cells treated with Col-Im complex. In the presence of periplasmic extracts, in vitro processing of DNase domain of ColE7 was also observed. These results suggest that the processing of ColE7 has occurred for translocation of the DNase-type colicin across the membrane and the process is probably taking place in the periplasmic space of the membrane.

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245.

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.

Direct antigen capture by soluble scFv antibodies. A method for detection, characterization, and determination of affinity.

For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.

Identification of an antigenic protein Pga30 from Porphyromonas gingivalis W50

Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that has been implicated as a major pathogen in the development of adult periodontitis. In an approach to identify a P. gingivalis antigen uniquely seroreactive with healthy subjects, we produced a surface and periplasmic extract of P. gingivalis, separated that extract into 36 fractions using anion-exchange chromatography and screened each fraction for reactivity in an enzyme-linked immunosorbent assay (ELISA) using sera from eight periodontitis patients and eight age- and sex-matched healthy controls. All of the diseased subjects harboured subgingival P. gingivalis by DNA probe analysis and exhibited similar seroreactivity profiles to the anion exchange fractions. However, only two of the healthy subjects (C10 and C15) were seroreactive with the fractions. The highest reactivity for all the seropositive subjects was with the same anion-exchange fractions 13-15. The anion exchange fraction (14) with the highest seroreactivity was subjected to gel filtration chromatography, and fraction 22 from this chromatography exhibited the highest reactivity with all the seropositive subjects. However, fraction 27 was found to be uniquely seroreactive with healthy subject C10, as it was not recognized by sera from any of the diseased or the other healthy subjects. This fraction was further purified by reversed-phase high-pressure liquid chromatography and shown to contain a 30-kDa protein as determined by SDS-PAGE. Control subject C10 had no pocket depths greater than 3 mm and no sites that bled on gentle probing; however, P. gingivalis was detected in subgingival plaque samples at a level of 10(5)-10(6) cells per site in two of the ten sites sampled. This subject was also unusual in that he exhibited a seroreactivity profile similar to that of diseased subjects, which was not characteristic of the healthy control subjects. The unique reactivity of the 30-kDa antigen, designated Pga30, with subject C10 serum was confirmed in a Western blot with the purified antigen. N-Terminal sequence analysis of Pga30 produced a single, unambiguous protein sequence confirming the purity of the protein. A search of the database using the N-terminal sequence obtained did not reveal any significant sequence similarity. In conclusion, we have identified a P. gingivalis antigen that was uniquely reactive in an ELISA and Western blot with serum from a subject with no clinical signs of periodontitis who harbored P. gingivalis in subgingival plaque.

An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract

An efficient cell-free translation system was developed by removal of phosphatase localized in the periplasmic space, which hampers the translation reaction by hydrolyzing ATP. S30 extract was prepared from the spheroplast of Escherichia coli, and as much as 40% of ATP-hydrolysis activity of phosphatases could be easily removed by the spheroplast formation. The reaction period of translation using phosphatase-removed S30 extract could be prolonged and protein synthesis was enhanced by more than 30%.

Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB.

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

Periplasmic localization of a GroES homologue in Escherichia coli transformed with groESx cloned from Legionella-like endosymbiontsin Amoeba proteus

Escherichia coli MC4100 transformed with a groE homologous operon cloned from X-bacteria accumulated large amounts of the gene product when cultured at 30 or 37 °C. Heat shock for 10–30 min at 42 °C or ethanol (5%) shock for 2 h increased GroESx levels to about twice that in E. coli grown at 30 °C. The subcellular localization of GroESx in transformed E. coli was determined by several subcellular fractionation methods, by the analysis of extracted proteins in SDS polyacrylamide gels and by assays of marker enzymes. The GroESx protein was detected in both the periplasmic and cytoplasmic extracts and a large amount of the protein was accumulated in the periplasm. The GroEL protein and recombinant β-galactosidase were exclusively localized in the cytoplasmic fraction, eliminating the possibility that periplasmic GroESx might be due to simple overproduction. N-terminal amino acid sequencing confirmed that the protein resolved on a 2-D gel was GroESx. This work represents the first report of the periplasmic location of GroES homologues in E. coli.

Quantitative Measurement of Bitagged Recombinant Proteins Using an Immunometric Assay: Application to an Anti-Substance P Recombinant Antibody

We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer. In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb. A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference. The method was applied to the direct measurement, in periplasmic extracts, of the total and active fractions of an ScFv produced at different induction temperatures.

Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli

New E. coli vectors based on the pOPE/pSTE vector system [Gene 128 (1993) 97] were constructed to express a single-chain Fv antibody fragment (scFv), a scFv–streptavidin fusion protein and two disulfide bond-stabilized Fv antibody fragments (dsFvs) utilizing different side chain positions for disulfide stabilization. All of these constructs encoded fusion proteins carrying five C-terminal histidine residues preceded by an unpaired cysteine. The influence of this cysteine, which was originally introduced to allow the chemical modification of the fusion proteins, was assessed by exchanging the two amino acids CysIle in front of the carboxy terminal His-tag to SerHis in all constructs. Yield and antigen-binding activity of the antibody constructs were compared after standard lab-scale periplasmic expression in Escherichia coli. The removal of the unpaired cysteine resulted in a significant increase in antigen-binding activity of the crude periplasmic extracts. Further, a three–five fold increase of yield and a significantly improved purity were observed after immobilized metal affinity chromatography (IMAC) with all four constructs.

Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase.

The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.

The Product of the Rice myb7 Unspliced mRNA Dimerizes with the Maize Leucine Zipper Opaque2 and Stimulates Its Activity in a Transient Expression Assay

myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.

Single-step purification of recombinant human growth hormone (hGH) directly from bacterial osmotic shock fluids, for the purpose of (125)I-hGH preparation.

A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.

Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display

In antibody–ribosome–mRNA complex (ARM) ribosome display, stable complexes of nascent protein, mRNA and ribosomes are produced in a eukaryotic in vitro expression system, through coupled transcription and translation of DNA lacking a 3′ stop codon. Selection of the protein simultaneously captures the relevant mRNA, which is recovered as DNA by coupled reverse transcription–polymerase chain reaction (RT-PCR) performed on the intact complexes. Here, we describe the use of ARM display to select a specific human antibody fragment from a transgenic mouse library. The mice carry unrearranged gene segments of the human heavy (H) and κ light (L) chain loci, while the endogenous murine H and κ loci are functionally silenced; they respond to immunisation by production of fully human IgM antibodies. A library encoding human single-chain (sc) antibody (V H/K) fragments, in which V H domains and κ light chains were combined at random by PCR, was prepared from spleen cells of transgenic mice immunised with progesterone-bovine serum albumin (BSA). Library diversity was demonstrated by sequencing. Progesterone-binding fragments were selected over five cycles of ARM display and the selected DNA cloned and expressed in Escherichia coli. Soluble V H/K fragments obtained in periplasmic extracts had the same specificity as ribosome-bound V H/K, supporting the view that folding and specificity of the displayed and soluble proteins are equivalent. The affinity of the expressed V H/K was ∼10 −8 M. Sequencing showed that ARM display selected a single V H/V L combination (V H1-2, Vκ4-1) and rearrangement, with a few mutational differences between clones. Monoclonal antibodies against progesterone-BSA obtained from hybridomas were encoded by the same V H and V L segments and had similar properties to the fragments obtained in vitro. The combination of ribosome display and transgenic mouse technologies is a rapid means of generating fully human antibody fragments in vitro for expression and further manipulation.

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low Km values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower kcat/Km values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.