Cloning, periplasmic expression and purification of hGM-CSF (Human Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli

Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds. Although GM-CSF is glycosylated in its natural form, the glycosylation perhaps has not been involved in its biological function. GM-CSF stimulates the survival, proliferation, and differentiation of hem at opoietic progenitor cells and also enhances the functional properties of mature myeloid cells. GM-CSF is used as a therapeutic agent in various clinical cases such as neutropenia following chemotherapy, bone marrow transplantation, acute myeloid leukemia… In this study, we report the results on the cloning and expression of recombinant human GM-CSF in the periplasmic space of Escherichia coli. The hGM-CSF gene was amplified by polymerase chain reaction using two oligonucleotide primers containing NcoI and XhoI restriction sites. This DNA fragment was successfully cloned between the NcoI and Xho I sites of the plasmid pET-22b, in frame with the pelB signal peptide sequence. The expression vector pET-hGM was transformed into E. coli BL21(DE3) and the transformants were induced by IPTG and examined for hGM-CSF production. Periplasmic proteins were released by osmotic shock treatment. The expression of recombinant hGM-CSF was evaluated by SDS–PAGE in total, cytoplasmic and periplasmic fractions. The recombinant hGMCSF in periplasmic fraction was then subjected to ion exchange chromatography using Q Sepharose FF column with saltincrement elution step. SDS-PAGE showed there was a visible expression of recombinant hGM-CSF in the periplasmic fraction of the E. coli BL21(DE3)/pET-hGM and a purified band with the purity of 97.4% after ion exchange chromatography. This result was further confirmed by Western blot using anti-hGM-CSF antibody.

Purification and characterization of azurin from the methylamine-utilizing obligate methylotroph Methylobacillus flagellatus KT

Methylamine dehydrogenase (MADH) and azurin were purified from the periplasmic fraction of the methylamine-grown obligate methylotroph Methylobacillus flagellatus KT. The molecular mass of the purified azurin was 16.3 kDa, as measured by SDS–PAGE, or 13 920 Da as determined by MALDI–TOF mass spectrometry. Azurin of M. flagellatus KT contained 1 copper atom per molecule and had an absorption maximum at 620 nm in the oxidized state. The redox potential of azurin measured at pH 7.0 by square-wave voltammetry was +275 mV versus normal hydrogen electrode. MADH reduced azurin in the presence of methylamine, indicating that this cupredoxin is likely to be the physiological electron acceptor for MADH in the electron transport chain of the methylotroph. A scheme of electron transport functioning in M. flagellatus KТ during methylamine oxidation is proposed.

Tat Pathway-Mediated Translocation of the Sec Pathway Substrate Protein MexA, an Inner Membrane Component of the MexAB-OprM Xenobiotic Extrusion Pump in Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is equipped with the Sec and Tat protein secretion systems, which translocate the xenobiotic transporter MexAB-OprM and the pathogenic factor phospholipase C (PlcH), respectively. When the signal sequence of MexA was replaced with that of PlcH, the hybrid protein was successfully expressed and recovered from the periplasmic fraction, suggesting that the hybrid protein had been translocated across the inner membrane. MexA-deficient cells harboring the plasmid carrying the plcH-mexA fusion gene showed antibiotic resistance comparable to that of the wild-type cells. This result suggested that MexA secreted via the Tat machinery was properly assembled and functioned as a subunit of the MexAB-OprM efflux pump. A mutation was introduced into the chromosomal tatC gene encoding an inner membrane component of the Tat protein secretion machinery in mexA-deficient cells, and they were transformed with the plasmid carrying the plcH-mexA fusion gene. The transformants showed antibiotic susceptibility comparable to that of mexA-deficient cells, indicating that the hybrid protein was not transported to the periplasm. Whole-cell lysate of the mexA-tatC double mutant harboring the plcH-mexA plasmid produced mainly unprocessed PlcH-MexA. The periplasmic fraction showed no detectable anti-MexA antibody-reactive material. On the basis of these results, we concluded that MexA could be translocated across the inner membrane through the Tat pathway and assembled with its cognate partners, MexB and OprM, and that this complex machinery was fully functional. This hybrid protein translocation system has the potential to be a powerful screening tool for antimicrobial agents targeting the Tat system, which is not present in mammalian cells.

Constitutive High Level Expression of an Endoxylanase Gene from the Newly IsolatedBacillus subtilisAQ1 inEscherichia coli

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted asBacillus subtilis(B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed inE. coli. InE. colithe recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely,2173.6 ± 51.4and2745.3 ± 11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the nativeB. subtilisAQ1 endoxylanase and that of 95% homologous recombinant one fromB. subtilisDB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression inE. coliand hence made the production inE. colifeasible.

A Preliminary Differential Proteomic Analysis of the Periplasmic Fraction in Acidithiobacillus Ferrooxidans Planktonic and Attached Cells Exposed to Chalcopyrite

The Acidithiobacillus ferrooxidans periplasmic space is known to have proteins involved in the respiratory chains. There are no reports about the expression of the periplasmic proteins in A. ferrooxidans cells attached to chalcopyrite. In this preliminary work, it was compared the periplasmic protein profiles of A. ferrooxidans planktonic and attached cells after exposure to chalcopyrite for 2 hours. The bacterial response to chalcopyrite was investigated by a proteomic approach (two- dimensional gel electrophoresis and mass spectrometry). Four proteins differentially expressed between planktonic and attached cells after exposure to chalcopyrite were identified. Two of these proteins, repressed in chalcopyrite- attached cells, were both identified as superoxide dismutase, whereas the single strand binding protein (SSB) and the PspA/IM30 protein were induced. These results showed that A. ferrooxidans chalcopyrite- attached and planktonic cells show differential expression of the periplasmic proteins and that a proteomic approach can provide a valuable tool to detect proteins related to the A. ferrooxidans response to attachment to the mineral substrates.

Shiga Toxin 2 Is Specifically Released from Bacterial Cells by Two Different Mechanisms

ABSTRACT Shiga toxin 1 (Stx1) is located in the periplasmic fraction, while Stx2 is found in the extracellular fraction, suggesting that enterohemorrhagic Escherichia coli (EHEC) contains a specific Stx2 release system. Both stx 1 and stx 2 are found within the late operons of Stx-encoding phages. Stx2 production is greatly induced by mitomycin C, suggesting that stx 2 can transcribe from the late phage promoter of the Stx2-encoding phage. However, the Stx1 promoter adjacent to stx 1 is a dominant regulatory element in Stx1 production. With the deletion of phage lysis genes of the Stx2-encoding phage, Stx2 remains in the bacterial cells. Further, we demonstrate that the Stx2-encoding phage, but not the Stx1-encoding phage, is spontaneously induced at extremely low rates. These results indicate that spontaneously specific Stx2-encoding phage induction is involved in specific Stx2 release from bacterial cells. Furthermore, to examine whether another system for specific Stx2 release is present in EHEC, we analyze the stx-replaced mutants. As expected, Stx2 derived from the Stx1 promoter is located in both the extracellular and cell-associated fractions, while mutant Stx2 (B subunit, S31N) derived from the Stx1 promoter is found only in the cell-associated fraction. These results indicate that EHEC has another Stx2 release system that strictly recognizes the serine 31 residue of the B subunit. Overall, we present evidence that specific Stx2 release from bacterial cells is involved in both the Stx2-encoding phage induction system and another Stx2 release system.

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

ABSTRACT l-(−)-Azetidine-2-carboxylate (AC) is a toxic, natural product analog of l-proline. This study revealed the genes and biochemical strategy employed by Pseudomonas sp. strain A2C to detoxify and assimilate AC as its sole nitrogen source. The gene region from Pseudomonas sp. strain A2C required for detoxification was cloned into Escherichia coli and sequenced. The 7.0-kb region contained eight identifiable genes. Four encoded putative transporters or permeases for γ-amino acids or drugs. Another gene encoded a homolog of 2-haloacid dehalogenase (HAD). The encoded protein, denoted l-azetidine-2-carboxylate hydrolase (AC hydrolase), was highly overexpressed by subcloning. The AC hydrolase was shown to catalyze azetidine ring opening with the production of 2-hydroxy-4-aminobutyrate. AC hydrolase was further demonstrated to be a new hydrolytic member of the HAD superfamily by showing loss of activity upon changing aspartate-12, the conserved active site nucleophile in this family, to an alanine residue. The presence of a gene encoding a potential export chaperone protein, CsaA, adjacent to the AC hydrolase gene suggested that AC hydrolase might be found inside the periplasm in the native Pseudomonas strain. Periplasmic and cytoplasmic cell fractions from Pseudomonas sp. strain A2C were prepared. A higher specific activity for AC hydrolysis was found in the periplasmic fraction. Protein mass spectrometry further identified AC hydrolase and known periplasmic marker proteins in the periplasmic fraction. A model was proposed in which AC is hydrolyzed in the periplasm and the product of that reaction is transported into and further metabolized in the cytoplasm.

Characterization of abn2 (yxiA), Encoding a Bacillus subtilis GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation

ABSTRACT The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-α-1,5-l-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-α-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent Km of 2.0 mg ml−1 and V max of 0.25 mmol min−1 mg−1 at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a σA-dependent and a σH-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.

Two Different Types of Dehalogenases, LinA and LinB, Involved in γ-Hexachlorocyclohexane Degradation inSphingomonas paucimobilis UT26 Are Localized in the Periplasmic Space without Molecular Processing

ABSTRACT γ-Hexachlorocyclohexane (γ-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a γ-HCH-degrading bacterium, Sphingomonas paucimobilis UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (γ-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of γ-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.

Functional Analysis of the Two-Gene Lysis System of the Pneumococcal Phage Cp-1 in Homologous and Heterologous Host Cells

ABSTRACT The two lysis genes cph1 and cpl1 of theStreptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed inEscherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressingE. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 andcpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.