Abstract
In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type l signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pl of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
| Language | English |
|---|---|
| Pages | 1230-1238 |
| Number of pages | 9 |
| Journal | Journal of Bacteriology |
| Volume | 179 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 1 Jan 1997 |
| Externally published | Yes |
ASJC Scopus subject areas
- Microbiology
- Molecular Biology
Cite this
}
Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2). / Champion, Cheryl I.; Blanco, David R.; Exner, Maurice M.; Erdjument-Bromage, Hediye; Hancock, Robert; Tempst, Paul; Miller, James N.; Lovett, Michael A.
In: Journal of Bacteriology, Vol. 179, No. 4, 01.01.1997, p. 1230-1238.Research output: Contribution to journal › Article
TY - JOUR
T1 - Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2)
AU - Champion, Cheryl I.
AU - Blanco, David R.
AU - Exner, Maurice M.
AU - Erdjument-Bromage, Hediye
AU - Hancock, Robert
AU - Tempst, Paul
AU - Miller, James N.
AU - Lovett, Michael A.
PY - 1997/1/1
Y1 - 1997/1/1
N2 - In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type l signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pl of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
AB - In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type l signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pl of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
UR - http://www.scopus.com/inward/record.url?scp=0031039160&partnerID=8YFLogxK
U2 - 10.1128/jb.179.4.1230-1238.1997
DO - 10.1128/jb.179.4.1230-1238.1997
M3 - Article
VL - 179
SP - 1230
EP - 1238
JO - Journal of Bacteriology
T2 - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 4
ER -