doi:10.1016/j.pep.2008.01.015
Copyright © 2008 Elsevier Inc. All rights reserved.
Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium
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Ebert Seixas Hannaa, Maria-Cristina Roque-Barreiraa, Guilherme Martines Teixeira Mendesd, Sandro Gomes Soaresa, b and Marcelo Brocchia, c, 
, 
aDepartamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil
bInvent Biotecnologia Ltda. ME, Incubadora Supera, Rua dos Técnicos s/n, Universidade de São Paulo, Ribeirão Preto, SP 14040-900, Brazil
cDepartmento de Microbiologia e Imunologia, Instituto de Biologia, Rua Charles Darwin s/n, UNICAMP, Campinas, SP 13083-862, Brazil
dDepartamento de Clínica Médica, Faculdade de Ciências Médicas, Rua: Tessália Vieira de Camargo, 126, UNICAMP, Campinas, SP 13083-887, Brazil
Received 19 November 2007;
revised 15 January 2008.
Available online 5 February 2008.
Abstract
Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation.
Keywords: Recombinant Dps; Glycosylation; Salmonella
Fig. 1. Construction of the recombinant vector containing the cassette for Dps expression. The open reading frame of the dps gene was amplified by PCR, including the 381 and 383-bp regions up and downstream, respectively. A pair of primers was designed to contain the SmaI and SalI restriction sites. The amplicon fragment was cloned into the PCR cloning vector pGEM®-T Easy. On the top right, the agarose gel electrophoresis of SmaI/SalI-digested recombinant vector showing the 1.2-kb excised fragment, together with the 3.0-kb fragment from the vector.
Fig. 2. SDS–PAGE analysis of Dps from S. enterica cells and construction of the S. enterica recA-null mutant. (A) Overnight cultures (3.0 ml) was pelleted, resuspended in reducing loading buffer and submitted to SDS–PAGE 12.5%. Gel was stained with Coomassie brilliant blue R250. First lane: lysate of transformed bacteria cells [S. enterica UK-1 (pGEM-T/dps)] over-expressing Dps (arrow); second lane: lysate of native S. enterica UK-1 cells. Molecular mass markers are recombinant proteins from full-range Rainbow (GE Healthcare); (B) Agarose gel electrophoresis (1%) demonstrating the deletion of the recA gene in S. enterica UK-1 strain. The left lanes are the results of PCR amplification using the wild-type strain. The right lanes exhibit the results with the mutant strains. Second and third lanes of both strains are the electrophoresis profile of PCR using primers specific for the cat gene amplification, which is negative for the wild-type strain and positive for the mutant strain.
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Fig. 3. Expression, purification, staining and immunoblot of recombinant Dps from S. enterica Typhimurium. Pur1, as for the first step of chromatography: FLA-3000 scanning of the Superdex 200 column fractions containing Dps, stained with Sypro® Ruby for proteins; Pur2, as for the second step of chromatography: FLA-3000 scanning of the jacalin purified fraction, also stained with Sypro® Ruby; gp (glycoprotein staining): UV-transilluminator photography of the jacalin purified fraction stained for glycoproteins with ProQ™ Emerald 300; W1 and W2 (Western blots): immunoblot of the gel filtration and jacalin-purified fractions, respectively, reacted with anti-Dps antibody. The excitation wavelength for FLA-3000 was 450 nm and scanned at 610 nm (Sypro® Ruby). The excitation wavelength for transilluminator was 280 nm, with maximum emission at 530 nm (ProQ™ Emerald 300). Molecular markers are from CandyCane glycoprotein molecular weight standards (included in the Molecular Probes staining kit—Invitrogen) for fluorescence and from Rainbow (GE Healthcare) for immunoblotting.
Table 1.
Purification yields of Dps from S. enterica UK-1 cell lysatesa
a 1.06 g of weight cells were used.
b The purity of recombinant Dps was estimated from protein band intensity in SDS–PAGE.
Corresponding author. Address: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil. Fax: +55 19 3788 6276.
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