Expression ofChlamydia psittaci-encoded polypeptides inEscherichia coli

Kihlström, Erik; Bogosian, Gregg; Bassford, P J; Wyrick, Priscilla B.

doi:10.1007/bf01589384

Cited by 3 publications

(1 citation statement)

References 17 publications

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“…Chiamydial DNA and plasmid preparation. Approximately 0.5 x 1010 to 5 x 1010 purified EB were used to isolate chlamydial genomic DNA as described previously (22). Generally, the purified genomic DNA fragments were greater than 50 kb in length, as determined by electrophoresis in a 0.3% agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Recombinant Escherichia coli clones expressing Chlamydia trachomatis gene products attach to human endometrial epithelial cells

et al. 1991

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To identify Chlamydia trachomatis genes involved in attachment to host cells, a chlamydial genomic library was screened on the basis of binding characteristics by two methods. In the whole-cell screen, individual recombinant Escherichia coli clones were assayed for adherence to eukaryotic cells. In the membrane-binding screen, each recombinant colony of E. coli was treated with CHCI3 and assayed for binding to purified, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized, 35S-labeled eukaryotic membrane material. Initial screening with McCoy ceUs was refined by using HEC-1B cells, a human endometrial epithelial cell line, which discriminate among recombinants adhering to McCoy cells. Some recombinants demonstrate significantly greater adherence to HEC-1B cells than to McCoy cells and appear, by transmission electron microscopy, to associate with electron-dense areas of the epithelial cell plasma membrane, resembling coated pits. Recombinants positive by one or both screening methods were examined by Southern and Western (immunoblot) analyses, which revealed the presence of chlamydial sequences inserted in the plasmids and the expression of novel 18-, 28-, and-82-kDa proteins, recognized by antichlamydial sera. Maxicell analysis of selected recombinants confirmed that the proteins of 28 and-82 kDa, and perhaps of 18 kDa, are plasmid encoded. Antiserum generated against the recombinant-82-kDa protein reacted in Western analysis with a similar-sized protein from C. trachomatis serovar E elementary bodies (EB) and reticulate * Corresponding author.

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Section: Methodsmentioning

confidence: 99%