The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins

Kühn, Sabine; Zipfel, Peter F.

doi:10.1016/0378-1119(95)00360-i

Cited by 111 publications

(98 citation statements)

References 10 publications

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“…Recombinant Lpd was sequenced by MALDI-TOF-MS/MS. Recombinant fragments of Factor H (SCRs 1-4, 1-5, 1-6, 8-11, 11-15, 15-18, 15-20, 18-20, and 19-20) and FHL-1 (SCRs 1-7) were expressed in the baculovirus system, as described (26). Recombinant CFHR1 and fragments of CFHR1 (SCRs 1-2, 3-4) were expressed in Pichia pastoris, as described (27).…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

Hallström

Mörgelin

Barthel

et al. 2012

The Journal of Immunology

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The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18–20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

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Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

Hallström

Mörgelin

Barthel

et al. 2012

The Journal of Immunology

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“…The FITC-conjugated goat anti-rabbit pAb was obtained from Dakopatts. Recombinant fragments of FH (SCRs 1-5, 1-6, 8 -11, 8 -20, 15-20, 18 -20, and 19 -20) and FHL-1 (SCRs 1-7) were expressed in the baculovirus system as described (36). Briefly, Sodoptera frugiperda (Sf9) cells were grown in expression medium (BioWhittaker) supplemented with streptomycin (100 g/ml), penicillin (100 U/ml), and amphotericin B (250 ng/ml) and infected with a recombinant baculovirus at a multiplicity of infection of 5.…”

Section: Proteins and Absmentioning

confidence: 99%

“…Briefly, Sodoptera frugiperda (Sf9) cells were grown in expression medium (BioWhittaker) supplemented with streptomycin (100 g/ml), penicillin (100 U/ml), and amphotericin B (250 ng/ml) and infected with a recombinant baculovirus at a multiplicity of infection of 5. The culture supernatants were collected 9 days after infection, and recombinant proteins were purified by Ni 2ϩ -chelate chromatography as described (36). The proteins were concentrated using Centricon microconcentrators with a cutoff at 10 kDa (Millipore).…”

Section: Proteins and Absmentioning

confidence: 99%

Haemophilus influenzae Interacts with the Human Complement Inhibitor Factor H

Hallström

Zipfel

Blom

et al. 2008

The Journal of Immunology

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Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6–7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18–20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of ∼32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.

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“…Cloning, expression in the baculovirus system, and purification of most of the used recombinant fragments of fH have been described earlier (27,36,50). Also the additional fragments representing SCR8-11, SCR15-18 and SCR15-20 were generated using the pBSV-8His expression vector (50).…”

Section: Recombinant Fragments Of Fhmentioning

confidence: 99%

“…Also the additional fragments representing SCR8-11, SCR15-18 and SCR15-20 were generated using the pBSV-8His expression vector (50). For amplification of cDNA fragments of After these initial steps VBS or 1/3 VBS was used as the continuous flow buffer at 5 µl/min; only degassed buffers were used.…”

Section: Recombinant Fragments Of Fhmentioning

confidence: 99%

Each of the Three Binding Sites on Complement Factor H Interacts with a Distinct Site on C3b

Jokiranta¹,

Hellwage²,

Koistinen³

et al. 2000

Journal of Biological Chemistry

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The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins

Cited by 111 publications

References 10 publications

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

Haemophilus influenzae Interacts with the Human Complement Inhibitor Factor H

Each of the Three Binding Sites on Complement Factor H Interacts with a Distinct Site on C3b

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