Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli

Abstract

This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.

1 Introduction

Actinobacillus pleuropneumoniae is a Gram-negative bacterium, a member of the family Pastereurellaceae, and is the ethiological agent of porcine pleuropneumonia, an acute or chronic respiratory infection affecting pigs of all ages. Epidemiological data suggest, however, that virulence of this bacterium is strongly correlated with the presence of Apx toxins [1]. Discoveries on the immunogenicity of A. pleuropneumoniae Apx toxins in infected pigs have stimulated studies trying to achieve protection from A. pleuropneumoniae by use of these toxins. Results of the studies using various combinations of exotoxins and membrane proteins [2] have shown the vaccine containing Apx I, Apx II, and Apx III toxins to provide most efficient protection from infection with all A. pleuropneumoniae serotypes [3]. Detailed studies of the clinical symptoms of the disease and its characteristic lung lesions, its experimental induction in pigs with viable and sonicated A. pleuropneumoniae, and the endobronchial inoculation of Apx toxins exist [4,5]. The virulence of A. pleuropneumoniae may be considered multifactorial, as is the case with most pathogenic bacteria; the factors involved in pathogenesis include capsular polysaccharides [6], lipopolysaccharides [7], membrane proteins [8,9], adhesion factors [10], exotoxins [5], and urease [11]. Epidemiological data suggest, however, that virulence is strongly correlated with the presence of Apx toxins [4,17].

RTX (repeat in the structural toxins) toxins are a class of pore-forming, cytolytic protein toxins that occur widely among pathogenic Gram-negative bacteria. RTX toxins appear to play a direct role in the virulence of A. pleuropneumoniae. These exotoxins are called Apx I, Apx II, and Apx III.

Discoveries on the immunogenicity of A. pleuropneumoniae Apx toxins in infected pigs have stimulated studies trying to achieve protection from A. pleuropneumoniae by use of these toxins. Results of the studies using various combinations of exotoxins and membrane proteins [2] have shown the vaccine containing Apx I, Apx II, and Apx III toxins to provide most efficient protection from infection with all A. pleuropneumoniae serotypes [3].

In this work, we describe the expression and purification of a histidine-tagged form of the Apx I coding sequence in Escherichia coli BL21(DE3)pLysS. Apx I is proposed for the RTX toxins produced by the reference strains for serotypes 1, 5a, 5b, 9, 10, 11, which was previously named hemolysin I (Hly I) or cytolysin I (Cly I). This protein is strongly hemolytic and shows strong cytotoxic activity toward pig alveolar macrophages and neutrophils [12].

The whole procedure involves a unique combination of the appropriate expression vector, bacteria host cell line, optimized growth conditions and purification of produced recombinant protein for further immunological studies.

2 Materials and methods

2.1 Strains and plasmids

E. coli strains TOP10F′ and BL21(DE3)pLysS were purchased from Invitrogen (The Netherlands). The pCRT7/NT TOPO vector has been described previously [13]. E. coli manipulations were performed according to the manufacturer's instructions [14]. Standard DNA and protein manipulations were carried out as described in Sambrook et al. [15] and Ausubel et al. [16].

2.2 Construction of bacterial expression vector

The coding region of A. pleuropneumoniae Apx I toxin [17] (GenBank Accession No. AF363362) was amplified by polymerase chain reaction (PCR) using Apx I AR (5′-atg gct aac tct cag ctc ga-3′) and Apx I AF (5′-tta agc aga ttg tgt taa at-3′) set of primers.

The PCR product, 3069 bp long, was purified using a PCR purification kit (Qiagen, Germany) and cloned into the pCRT7/NT TOPO E. coli expression vector (Invitrogen), creating pCRT7/NT:Apx I in which protein expression is under control of the bacteriophage T7 promoter. The recombinant DNA was transformed into chemically competent E. coli TOP10F′ cells which were used for propagation of plasmid construct.

The transformants were selected on Luria–Bertani (LB) plates containing 100 μg ml⁻¹ ampicillin. Mini-scale isolation of plasmid DNA was used for the preparation of recombinant plasmid for sequencing. The presence of an open reading frame (ORF) was confirmed by PCR screening of recombinant plasmid and by sequencing. For the PCR screening, Apx I AF, Apx I AR, T7 F and T7 R primers (GenBank Accession Numbers AA113728 and AJ440790) were used. The sequencing reactions were performed in an automated DNA sequencer (Abi Prism 310 Genetic Analyzer, Applied Biosystems, USA). Sequencing data were assembled and edited by the BLAST method.

2.3 Tranformation of E. coli expression cells

Once the sequence of pCRT7/NT:Apx I was verified by sequencing and PCR screening, the construct was transformed into BL21(DE3)pLysS host cells (Invitrogen) which contain T7 lysozyme to degrade any background T7 RNA polymerase formed. The cell line contains an introduced bacteriophage T7 RNA polymerase gene under lacUV5 control (DE3 lysogen). Therefore, both T7 RNA polymerase and the cloned insert are normally repressed by the laqI gene repressor protein until induction by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG).

Positive clones were grown on LB plates with 100 μg ml⁻¹ ampicillin and 34 μg ml⁻¹ chloramphenicol at 37°C for 24 h. A single colony from the culture was grown in LB medium (10 g l⁻¹ bacto-tryptone, 5 g l⁻¹ yeast extract, and 10 g l⁻¹ NaCl, pH 7.0) with 100 μg ml⁻¹ ampicillin and 34 μg ml⁻¹ chloramphenicol to an optical density of 0.6–0.7, protein expression was induced by the addition of 100 mM IPTG with further growth at 37°C for 4 h. Cells were harvested by centrifugation (10 min, 4000×g) and cells and cell culture supernatant samples were examined by Coomassie brillant blue R250-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel and by Western blot assay. The clones producing the protein of interest were identified by the observation of a band of the appropriate size (105 kDa).

2.4 Purification of Apx I expressed by E. coli

To purify Apx I, bacterial cells under the conditions described above were pelleted, and twice washed in PBS (150 mM NaCl, 10 mM NaH₂PO₄, pH 8.0). The cells were lysed by the addition of 0.1 mg ml⁻¹ lysozyme at room temperature for 60 min. The lysed cells were then sonicated using an Ultrasonic Cell Disruptor (Misonix, USA). After the sonication step (12°C, 5 min), the cells were heat-shocked (cells were frozen at −80°C for 30 min, then incubated at 37°C for 10 min) three times. After each heating step the sonication was included.

The recombinant protein was purified by use of the 6× His affinity tag that has a very high affinity to nickel-nitrilotriacetic acid (Ni-NTA agarose and Ni-NTA superflow, Qiagen, USA). The inclusion bodies prepared from 1 l of cell culture were solubilized in 40 ml of denaturation buffer (6 M guanidium hydrochloride, 0.1 M Tris, pH 8.6) whereafter the lysate was centrifuged (20 min, 10 000×g). Ni-NTA resin was added (4 ml) and after an incubation period of 1 h at room temperature, the resin was collected by centrifugation (5 min, 800×g) and washed three times with 10 ml of denaturation buffer. The recombinant protein was eluted by addition of 5 ml denaturation buffer +0.5 M imidazole. After the incubation period of 5 min at room temperature the suspension was centrifuged (5 min, 800×g) and the supernatant was analyzed by SDS–PAGE and by Western blotting analysis.

2.5 SDS–PAGE and Western blotting analysis

SDS–PAGE analysis was performed on 8% polyacrylamide gels under denaturing conditions. Gels were stained with Coomassie brillant blue R250. The method was used according to Sambrook et al. [15]. For Western blotting analysis, proteins were first separated on 8% gels and then transferred onto a PVDF membrane (Millipore) using 25 mM Tris, 190 mM glycine, and 15% methanol. Blots were incubated first with anti-Xpress antibody (Invitrogen) followed by a mouse peroxidase conjugated antibody (Sigma, USA). Proteins were visualized using a 3,3′-diaminobenzidine tetrahydrochloride (Fluka, Sweden) according to the manufacturer's instructions.

2.6 HPLC running conditions

High-performance liquid chromatography (HPLC) separations were carried out on a Ni-NTA agarose superflow (Novagen) Peek column (4.5×150 mm). The mobile phase used at 0.5 ml min⁻¹ flow rate for equilibration was 6 M guanidium hydrochloride and 0.1 M Tris, pH 8.6. For the elution, equilibration buffer supplemented with 0.5 M imidazole was used. The absorbance was measured using an UV LCD 2084 detector (Ecom, Czech Republic) at λ 280 nm. The main peak was manually collected as a 2-ml fraction. The Ni-NTA agarose was re-charged using 50 mM nickel sulfate.

3 Results and discussion

Apx I is one of the virulence factors of A. pleuropneumoniae, a bacterial species which is the causative agent of porcine pleuropneumonia. To study the role of Apx I in pathological processes, large amounts of Apx I have to be produced for the further study of Apx I biological activity.

A DNA sequence encoding the Apx I amino acid sequence was cloned under the control of the E. coli T7 promoter to produce the plasmid pCRT7/NT TOPO:Apx I. The resulting vector (pCRT7/NT TOPO:Apx I) has an ORF coding for the Apx I, an Xpress epitope for antibody detection of recombinant protein and a tag of six consecutive histidine residues (6× His-tag) on the N-terminus. The 6× His-tag is used like a potential purification aid.

The nucleotide sequence of the Apx I coding region of the pCRT7/NT TOPO:Apx I plasmid was confirmed by DNA sequencing and by PCR screening using the Apx I AF, Apx I AR, T7 F and T7 R primers.

The pCRT7/NT TOPO:Apx I plasmid was introduced first into the chemically competent E. coli TOP10F′ cells and after the propagation and maintenance of the construct into E. coli BL21 (DE3)pLysS cells. The ampicillin/chloramphenicol-resistant transformants were selected. Typically, efficiencies of 10⁷–10⁹ transformants per μg can be achieved depending on the strain of E. coli and the method used [18]. There are many enzymatic activities in E. coli which can destroy incoming DNA from non-homologous sources and reduce the transformation efficiency. Large DNA transforms less efficiently, on a molar basis, than small DNA. In our study, when transforming large 3069 bp DNA, a total of 25 transformants were obtained and five of these were selected for small-scale expression studies.

3.1 Small-scale expression and time course studies

Small-scale (10 ml) expression studies of five of the isolates described above were used to select the best Apx I-producing isolates. Cells were grown in LB medium as described in Section 2 and expression was induced by IPTG. Expression is under the control of the strong T7 promoter and is induced by IPTG. Time course expression studies were performed and aliquots taken at 0, 1, 2, 3 and 4 h after IPTG induction. Cell fractions and cell culture supernatants were analyzed for Apx I production by SDS–PAGE and by Western blotting using an anti-Xpress antibody. In the bands containing samples of cultured cells the clear evidence of production of a 105-kDa protein was seen on the SDS–PAGE gel. The protein was recognized by an anti-Xpress antibody when blotted onto a PVDF membrane. This band was absent in a vector-only control (data not shown).

Coomassie-stained SDS–PAGE analysis of the five clones showed that two of them produced a band corresponding to the predicted 105-kDa molecular mass of Apx I. Both of these clones produced similar amounts of the Apx I (data not shown), and one clone designated E. coli BL21 (pCRT7/NT TOPO-Apx I)-24 was chosen at random for all further work.

Fig. 1 shows the levels of A. pleuropneumoniae Apx I toxin protein that were generated in E. coli BL21 (pCRT7/NT TOPO-Apx I)-24 host cells, averaging 2–3 mg l⁻¹ of bacterial cells. Apx I was found predominantly in the insoluble fraction.

3.2 Purification of Apx I

The affinity of the His-tag for divalent metal ions was used to bind the engineered complex to a Ni²⁺-nitrilotriacetic acid commercial resin and allows an easy recovery of Apx I. Because Apx I expression resulted in insoluble inclusion bodies, the first purification step was the cell disruption and the inclusion bodies' isolation.

The results from the purification of Apx I using HPLC are shown in Fig. 2. To determine the chromatographic conditions, after solubilization of the inclusion bodies in 6 M guanidium hydrochloride, the solution was centrifuged at 10 000×g for 30 min and the supernatant was applied to a Ni-NTA column. Elution was achieved using 0.5 M imidazole. The recombinant protein was eluted as a single peak.

The main peak was manually collected as a 2-ml fraction (Fig. 2). The Ni-NTA agarose was re-charged using 50 mM nickel sulfate. The yield of HPLC-purified Apx I from 1 l of original culture is about 1.0–1.5 mg. The amount of protein was measured using Bicinchoninic Acid Protein Assay (Sigma, USA).

The amount and the purity of recombinant Apx I allows its use for either the preparation of vaccine against porcine pleuropneumonia (in combination with other Apx toxins) or the immunization of mice and rabbits for the specific antibody production and preparation of diagnostic pleuropneumonia tools.

References