Construction of His6-tagging vectors allowing single-step purification of GroES and other polypeptides produced in Bacillus subtilis

Schön, Ulrike; Schumann, Wolfgang

doi:10.1016/0378-1119(94)90044-2

Cited by 31 publications

(25 citation statements)

References 14 publications

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“…Accordingly, the yqfS gene was expressed in E. coli with a N-terminal hexahistidine tag and purified to apparent homogeneity by Ni-NTA-agarose chromatography. As has been described for other His-tagged proteins (21,25), His 6 -YqfS exhibited a slow migration on SDS-PAGE, showing a molecular mass of ϳ36 kDa, 3 kDa above its predicted mass.…”

Section: Discussionsupporting

confidence: 72%

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

et al. 2003

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The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His 6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His 6 -YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His 6 -YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3-phosphatases. However, the purified protein lacked both 5-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3 blocking groups of DNA generated during both spore dormancy and germination.During unpredicted periods of dormancy Bacillus subtilis spores are constantly exposed to environmental conditions that have the potential to cause several types of DNA damage. Therefore, the existence of spore-specific protecting mechanisms would seem to be fundamental for spore survival. One of the factors intricately involved in protecting spore DNA from several types of damage, such as oxidative stress, UV-C irradiation, and desiccation, is the presence of ␣/␤ type small acid-soluble proteins (reviewed in references 16, 28, and 27). Although ␣/␤ type small acid-soluble proteins protect spore DNA from several stresses, they confer protection neither to base alkylation (29) nor to UV-induced DNA strand break formation (30). Thus, while the physiological state of the B. subtilis spores prevents or dramatically slows DNA damage during the long periods of dormancy, it is clear that spores do accumulate potentially lethal and mutagenic DNA lesions such as the spore photoproduct, strand breaks, cyclobutane pyrimidine dimers, chemically altered bases and apurinic/apyridiminic (AP) sites which could affect transcription and replication processes during germination (16,26,29). To remove these potentially deleterious DNA damages and alteratio...

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Section: Discussionsupporting

confidence: 72%

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

et al. 2003

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“…The band (native GroES) showed a mol. wt slightly lower than that of recombinant GroES, in accordance with the observation that histidine-tagged proteins run more slowly than the equivalent, untagged proteins [27]. As HSPs are usually regarded as intracellular proteins involved in protein folding and assembly, in theory they should not be found outside the cells.…”

Section: Discussionsupporting

confidence: 79%

Recombinant GroES in combination with CpG oligodeoxynucleotides protects mice against Mycobacterium avium infection

Fattorini¹,

Creti²,

Nisini³

et al. 2002

Journal of Medical Microbiology

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“…The plasmids used in this study were pBC KS ϩ (Stratagene, La Jolla, Calif.), pQE30 (Qiagen), pUSH1 (45), and the pBluescript SK(Ϫ)-derived integrative vector for lactobacilli, pRV300 (26).…”

Section: Methodsmentioning

confidence: 99%

“…To overproduce HPr from L. casei, a DNA fragment carrying the L. casei ptsH gene with BamHI and HindIII restriction sites at the 5Ј and 3Ј ends, respectively, was amplified by PCR by using plasmid pVMH1 as template (51). After verifying the correct sequence, the L. casei ptsH gene was inserted into the shuttle vector pUSH1 (45). The resulting plasmid pLCHPr was transformed into E. coli ECE89 for overproduction of L. casei HPr as previously described (45).…”

Section: Methodsmentioning

confidence: 99%

“…After verifying the correct sequence, the L. casei ptsH gene was inserted into the shuttle vector pUSH1 (45). The resulting plasmid pLCHPr was transformed into E. coli ECE89 for overproduction of L. casei HPr as previously described (45). Since the Histagged HPr did not bind to Ni-nitrilotriacetic acid columns, the crude extract was kept for 10 min at 75°C, and precipitated proteins were removed by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

Dossonnet¹,

Monedero²,

Zagorec³

et al. 2000

J Bacteriol

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We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-SerHPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl ␤-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.Bacteria can respond to the presence of abundant carbon sources via a complex network of regulatory processes. Rapidly metabolizable carbohydrates such as glucose inhibit expression of catabolic operons encoding the enzymes necessary for the uptake and metabolism of less-favorable carbon sources (43) and also affect the synthesis of enzymes of central metabolic pathways such as glycolytic enzymes, as has been shown in Lactobacillus casei (31) and Lactococcus lactis (28). In grampositive bacteria, the metabolite-activated HPr kinase/P-SerHPr phosphatase (HprK/P) (2, 13, 22, 38) is the first enzyme of the signal transduction pathway used to control several of these regulatory processes. This bifunctional enzyme catalyzes the phosphorylation of HPr at the regulatory seryl residue 46 (5) as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) (22). HPr is a phosphocarrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). The PTS catalyzes the concomitant uptake and phosphorylation of carbohydrates by forming a protein phosphorylation cascade. HPr becomes intermediately phosphorylated by PEP and enzyme I at the catalytic His-15, and P-HisHPr transfers the phosphoryl group to the sugar-specific enzyme II complexes which transport and phosphorylate their corresponding substrates (35).Carbon catabolite repression (CCR) in gram-positive bacteria was found to be one of the P-Ser-HPr-controlled processes.

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Construction of His6-tagging vectors allowing single-step purification of GroES and other polypeptides produced in Bacillus subtilis

Cited by 31 publications

References 14 publications

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

Recombinant GroES in combination with CpG oligodeoxynucleotides protects mice against Mycobacterium avium infection

Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

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