Protocol for pseudomonas conjugation

Question

I'm trying to get a detailed protocol for mating E.coli (donor) with pseudomonas (recipient )..

Answer 1

Conjugating recombinant proteins to Pseudomonas aeruginosa ExoProtein A: A strategy for enhancing immunogenicity of malaria vaccine candidates

Abstract

Conjugation of polysaccharides to carrier proteins has been a successful approach for producing safe and effective vaccines. In an attempt to increase the immunogenicity of two malarial vaccine candidate proteins of Plasmodium falciparum, apical membrane antigen 1 (AMA1) to a blood stage vaccine candidate and surface protein 25 (Pfs25) a mosquito stage vaccine candidate, were each independently chemically conjugated to the mutant, nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA). AMA1 is a large (66 kD) relatively good immunogen in mice; Pfs25 is a poorly immunogenic protein when presented on alum to mice. Mice were immunized on days 0 and 28 with AMA1– or Pfs25–rEPA conjugates or unconjugated AMA1 or Pfs25, all formulated on Alhydrogel. Remarkably, sera from mice 14 days after the second immunization with Pfs25–rEPA conjugates displayed over a 1000-fold higher antibody titers as compared to unconjugated Pfs25. In contrast, AMA1 conjugated under the same conditions induced only a three-fold increase in antibody titers. When tested for functional activity, antibodies elicited by the AMA1–rEPA inhibited invasion of erythrocytes by blood-stage parasites and antibodies elicited by the Pfs25–rEPA conjugates blocked the development of the sexual stage parasites in the mosquito midgut. These results demonstrate that conjugation to rEPA induces a marked improvement in the antibody titer in mice for the poor immunogen (Pfs25) and for the larger protein (AMA1). These conjugates now need to be tested in humans to determine if mice are predictive of the response in humans.

Answer 2

There is a paper on the topic here

http://www.pubmedcentral.nih.gov/article...