Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae.

Zlotnick, Gary W.; Sanfilippo, V.; Mattler, J A; Kirkley, David H.; Boykins, Robert A.; Seid, Robert C.

doi:10.1016/s0021-9258(19)81587-9

Cited by 31 publications

(4 citation statements)

References 28 publications

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“…However, indirect evidence indicates that it performs a A db c_ -738 bp structural function for the cell. P6 is a lipoprotein and is associated with peptidoglycan (47,48). Furthermore, P6 shares homology with the peptidoglycan-associated lipoprotein of Escherichia coli (48).…”

Section: Discussionmentioning

confidence: 99%

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Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms

et al. 1991

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Infections caused by Haemophilus influenzae are a major worldwide health problem. In particular, nontypeable strains of H. influenzae are a common cause of otitis media in infants and children. A vaccine to prevent these infections would result in the prevention of substantial morbidity and cost savings. A problem in identifying an appropriate vaccine antigen has been the enormous antigenic heterogeneity among nontypeable strains of H. influenzae. The present study was undertaken to characterize the conservation of the P6 outer membrane protein (approximately 16,000 daltons) among strains of H. influenzae. A total of 20 type b strains and 20 nontypeable strains of diverse geographic and clinical origins was studied. Three approaches were taken. (i) Antigenic determinants recognized by monoclonal and polyclonal antibodies were present on P6 in all 40 strains tested. The molecular weight of P6 was identical in all strains. (ii) Comparison of the DNA sequences of the P6 genes from three epidemiologically and serologically unrelated strains demonstrated 100% homology at the amino acid level and 97 to 99% homology at the nucleotide level. (iii) Restriction fragment length polymorphism analysis demonstrated that the P6 gene and flanking sequences were highly conserved among all strains. These three independent series of experiments indicated that the P6 protein is highly conserved among strains of H. influenzae. P6 should receive serious consideration for inclusion in a vaccine to prevent infections caused by nontypeable H. influenzae.

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Section: Discussionmentioning

confidence: 99%

“…P6 is a lipoprotein and is associated with peptidoglycan (47,48). Furthermore, P6 shares homology with the peptidoglycan-associated lipoprotein of Escherichia coli (48). Therefore, it appears that P6 is the analog in H. influenzae of the peptidoglycan-associated lipoprotein in E. coli.…”

Section: Discussionmentioning

confidence: 99%

Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms

et al. 1991

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“…The mycoplasmal di-acylated lipopeptides lack the amide-bound lipid chain. Palmitoyl groups are the most common lipid chains in the bacterial lipoproteins but other lipid chains are also found (Belisle et al, 1994;Braun, 1975;Mizuno, 1979;Zlotnick et al, 1988). Synthetic lipopeptide analogs containing di-or tri-acylated cysteine groups mimic the proinflammatory properties of lipoproteins, thus confirming that the acylated N-terminal cysteine is the principal immune stimulatory motif (Berg et al, 1994;Bessler et al, 1985;Seifert et al, 1990;Wiesmu ¨ller et al, 1992).…”

Section: Introductionmentioning

confidence: 91%

Crystal Structure of the TLR1-TLR2 Heterodimer Induced by Binding of a Tri-Acylated Lipopeptide

Jin

Kim

Heo

et al. 2007

Cell

1,171

1,271

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TLR2 in association with TLR1 or TLR6 plays an important role in the innate immune response by recognizing microbial lipoproteins and lipopeptides. Here we present the crystal structures of the human TLR1-TLR2-lipopeptide complex and of the mouse TLR2-lipopeptide complex. Binding of the tri-acylated lipopeptide, Pam(3)CSK(4), induced the formation of an "m" shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam(2)CSK(4), did not. The three lipid chains of Pam(3)CSK(4) mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR1-TLR2 heterodimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.

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“…A well-known protocol for the solubilization and purification of a bacterial wild-type PAL was first developed in 1988 (Zlotnick et al, 1988). However, although that protocol uses several ultracentrifugation steps, heat treatment, and detergents, it results in a low protein yield and is not appropriate for recombinant PAL-enriched inclusion bodies that are produced in E. coli.…”

Section: Commentary Background Informationmentioning

confidence: 99%

Solubilization, Folding, and Purification of a Recombinant Peptidoglycan‐Associated Lipoprotein (PAL) Expressed in Escherichia coli

Santos

Souza

2018

CP Protein Science

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Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan-associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram-negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production. This protocol describes an efficient protein refolding method to solubilize and purify a recombinant PAL. Initially, recombinant PAL-enriched inclusion bodies obtained after the induction of PAL expression in Escherichia coli are treated with 8 M urea and then undergo buffer exchange via dialysis. Afterward, the soluble, recombinant PAL is purified using standard chromatographic methods. © 2018 by John Wiley & Sons, Inc.

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Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae.

Cited by 31 publications

References 28 publications

Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms

Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms

Crystal Structure of the TLR1-TLR2 Heterodimer Induced by Binding of a Tri-Acylated Lipopeptide

Solubilization, Folding, and Purification of a Recombinant Peptidoglycan‐Associated Lipoprotein (PAL) Expressed in Escherichia coli

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