|
|
|
|
|
|
Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli. K. J. Mukherjee, 2004.Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host . The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures . The expression system is based on two plasmids; one carries the product gene expressed from  PL under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from
PR . Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously . Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant . In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD600] of 3.5) was 37 mg liter–1, compared to a maximum of 13 mg liter–1 in the control culture (final OD600 of 20) . In a fed-batch fermentor culture, quiescent cells were held at an OD600 of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter–1 . A control culture with a similar feed reached an OD600 of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter–1 . Quiescent cells at an OD600 of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter–1 was achieved in 8 h . The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture . This study demonstrates that it is possible to establish high-density quiescent E . coli cultures that are capable of efficient synthesis, folding, and export of proteins .
Identification of Target Genes Regulated by the Two-Component System HP166-HP165 of Helicobacter pylori. Patricia Dietz, 2002.Two-component systems are signal transduction systems which enable bacteria to regulate cellular functions in response to changing environmental conditions . In most cases regulation is accomplished on the transcriptional level by a response regulator protein, which, according to the phosphorylation state of its receiver domain, displays different affinities for its target promoters . Here we describe identification of genes regulated by the two-component system HP166-HP165 of Helicobacter pylori and characterization of the corresponding target promoters . We demonstrated that expression of the HP166-HP165 two-component system is negatively autoregulated under conditions favoring autophosphorylation of the histidine kinase . Furthermore, we found that response regulator HP166 activates transcription of genes encoding a protein family with an unknown function present in H . pylori 26695, as well as an operon composed of five H . pylori-specific genes . While open reading frame HP166 is an essential gene, the target genes of the response regulator are not required for growth under in vitro culture conditions . Identification of the cAD1 Sex Pheromone Precursor in Enterococcus faecalis. Florence Y. An, 2002.The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci . The determinant for the pheromone in E . faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety . The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues . The nonisogenic E . faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8) . Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1 . The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response . A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential . A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor . Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety . Expression of spoT in Borrelia burgdorferi during Serum Starvation. Marc B. Concepcion, 2003.Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis . A 2.9-kb fragment containing a putative spoT gene was isolated from B . burgdorferi genomic DNA by PCR amplification and cloned into a pBAD24 vector . The cloned gene complemented Escherichia coli mutant strain CF1693, which contains deletions of both the relA and spoT genes . The spoT gene in E . coli encodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates the stringent response during carbon source starvation . B . burgdorferi has been reported to have a stress response to serum starvation . Thin-layer chromatography was used to detect (p)ppGpp extracted from H332PO4-labeled B . burgdorferi cells starved for serum in RPMI . B . burgdorferi spoT gene expression was characterized during fatty acid starvation . Northern analysis of spoT revealed detectable message at 2.5 min of starvation in RPMI . Expression of spoT during serum starvation increased  6-fold during the 30 min that starvation conditions were maintained . Further, expression of spoT decreased when serum was added to serum-starved cells . Reverse transcriptase PCR (RT-PCR) was used to detect spoT mRNA from
106 cells starved for serum in RPMI for 2.5 to 30 min or incubated in tick saliva for 15 min . Northern blot analysis suggests that spoT transcript was
900 nucleotides in length . RT-PCR amplification of the transcript using several sets of primers confirmed this finding . Additionally, a truncated clone containing only the first 950 bp of the 2,001-bp spoT open reading frame was able to complement E . coli CF1693 . The data suggest that B . burgdorferi exhibits a stringent response to serum starvation and during incubation in tick saliva .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
