Move over, E. coli. Stand aside, Vaccinia virus. Make way, CHOcells. Steer clear, yeast cultures.
Here comes a new kid on the host organism block. It starts itslife cycle as a single cell, and winds up (literally) as amulticellular slug. Meet Dictyostelium discoideum, (Dd) a soilamoeba, and (perhaps) future expression system.
Late last year, a European patent application (WO 92/20806)disclosure revealed efforts by a fledgling Swiss company,Dictagene SA, of this Lausanne suburb, to express a malarialparasite epitope in transformed Dd cells.
Dictagene's prime mover is cell biologist Christophe Reymond,who is on the faculty of Lausanne University. Reymond toldBioWorld that Dictagene "aims at developing potentials of Dd asan expression system for foreign proteins."
His patent application claims "a recombinant DNA moleculesuitable for the expression of a desired functional polypeptide,"including a Dd promoter region, leader sequence andtermination region. In particular, it claims a DNA encoding thecircum-sporoite (CS) antigen from Plasmodium falciparum, theprincipal malarial parasite, to make a malaria vaccine.
Reymond is one of a tiny handful of Dd researchers worldwidewho are trying to train Dictyostelium to take on recombinanttasks. They include: Arturo de Lozanne, Duke University,Durham, N.C.; Theo Dingermann, J.W. Goethe University,Frankfurt, Germany; William F. Loomis, University of California,San Diego; and Keith Williams, Macquarie University and theATIC Corp., Sydney, Australia.
Dictyostelium, Loomis tells BioWorld, derives from two Greekwords: "Dictyo" means network; "stelium," tower. Themillimeter-long amoebae typically crawl over rotting logs indamp woods, initially as free-living cells, feeding on bacteria,and dividing. But under harsh conditions, the starving cells,driven by spiral waves of chemoattraction, converge straighton a central point. There they coalesce, or aggregate, into amulticellular, slow-crawling, slug-like organism, which puts outspores to complete Dd's life cycle.
Scientists seeking a subunit malaria vaccine, Reymond pointsout, have cloned synthetic peptides containing theimmunodominant region of the CS protein in conventionalexpression systems, "but with limited success. The entire CSprotein," he observes, "could confer stronger, longer-lastingimmune protection." Such expression, Reymond's patent textnotes, has been obtained in vaccinia and baculovirus vectors,"but unstable protein expression and high cost of productionrender such systems unattractive."
To get around these drawbacks, he recruited the Dictyosteliumcell, which doubles every four hours, and reaches densities of1010 per liter.
As of February 1993, he told BioWorld, "the amount of CS anti-genic protein we have obtained is not extremely high; not themilligram per liter we expected."
But help is at hand. Birgit Wetterauer, a post-doctoral colleagueat the University of Munich's Zoological Institute, has suppliedthe Dictagene team with a mutant strain of Dd thatoverexpresses the discoidin promoter. "So genes placed underthe control of this wild-type promoter," Wetterauer toldBioWorld, "are over-expressed, which makes it suitable fordirecting over-expression of heterologous proteins."
Using Wetterauer's discoidin mutant, Reymond is now trying toboost gene CS expression, and in parallelH"which will make itmuch more interesting"Hobtain secretion in the medium. He isnow at the stage of doing the transformation, "and within a fewmonths we should have the boosted expression and secretion."
Progress and Setbacks
In Frankfurt, Theo Dingermann has expressed humanantithrombin III (AT III) in Dd, "in a reasonable amount," hetold BioWorld," though not terrific compared to E. coli orbacculovirus, or whatever," and secreted the entire protein.
"Unfortunately," Dingermann went on, "it is not correctlyglycoslylated." He is now planning to use different promoters,and switch from AT III to alpha-1-antitrypsin.
Cell biologist William F. Loomis, UC/San Diego, has studiedDictyostelium for 28 years. His most recent Dd-expressedprotein is the lethal chain of ricin.
"It certainly has a lot of potential as an expression system,"Loomis told BioWorld. "The way Dd makes and modifiesproteins is different from bacteria and yeast. Its mechanism issimpler than mammalian cells. On that basis alone, it isinteresting to explore. The system has yet to be proven, butthere are no insurmountable obstacles to its success, notaxonomic kingdom barrier. It's still a bit of an art to getexpression from proteins in any host cell."
Moreover, Loomis stated that "Dictyostelium has the essentialsfor commercial application: It's tamed; it grows well; it's ahaploid eukaryote, and can be treated in an industrial manner."
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.