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directed evolution of aspartate transaminasefor example. As reclon-ing of a library is laborious and prone to loss of diversity, and the effect
evolving highly efficient enzymes on selection pressure only qualitatively predictable, inducible systems
that provide a tightly controlled and graded transcriptional response toan external inducer represent potentially attractive alternatives.
Regulable promoters that use arabinoseor tetracyclias indu-
cer compounds have been well characterized. Both allow homo-
Caroline Heintz, Peter Kast & Donald Hilvert
geneous gene expression over a broad dynamic range. To constructselection plasmids that combine inducer dose-dependent gene expres-
Combining tunable transcription with an enzyme-degradation
sion with the convenience of high-copy plasmids, we opted for the
tag affords an effective means to reduce intracellular enzyme
tetracycline-inducible Ptet systemwhich does not require specifically
concentrations from high to very low levels. Such fine-tuned
engineered host strains. We replaced the weak, constitutive bla
control allows selection pressure to be systematically
promoter on our selection plasmid pKECMB(Fig. 1a) with a
increased in directed-evolution experiments. This facilitates
modified Ptet promoter cassette (Fig. 1b), which includes a down-
identification of mutants with wild-type activity, as shown
stream T7 promoter, to simplify protein overproduction of selected
here for an engineered chorismate mutase. Numerous selection
variants. As expected, auxotrophic KA12/pKIMP-UAUC cells harbor-
formats and cell-based screening methodologies may benefit
ing the gene for the very weakly active hEcCM under the control of
from the large dynamic range afforded by this easily
this promoter system (on plasmid pKT) did not grow under selective
conditions in the absence of inducer, but they regained prototrophy athigh tetracycline concentrations (Table 2). In contrast, cells containing
Genetic selection can greatly facilitate the search for rare catalysts in
the more active tEcCM and EcCM variants grew even in the absence of
very large protein libraries. Auxotrophic strains, which grow only if
inducer. Thus, background transcription affords sufficient amounts of
provided with a protein that functionally replaces a missing cellular
these more active catalysts to fully satisfy the metabolic needs of the
enzyme, are frequently used for this purpose. However, growth
cell. Clearly, in this case, tight transcriptional control alone is not
represents an indirect (and imperfect) readout of catalyst activity.
sufficient to reduce protein concentrations to a level low enough to
Even mediocre catalysts may provide sufficient activity for cells to
allow discrimination between a moderately active catalyst (tEcCM)
grow at wild-type levels, making it difficult to distinguish the most
active variants from their less effective counterparts. As a result, it can
To reduce protein concentration further, we fused an 11-amino-acid
be difficult or impossible to optimize relatively inefficient enzymes SsrA degradation signalto the C terminus of the catalyst (Fig. 1c).
through multiple rounds of mutagenesis and selection.
The SsrA tag targets the catalyst for rapid degradation by the
We faced this problem when we tried to improve an engineered
intracellular ClpXP protease. The efficacy of this strategy was estab-
chorismate mutase. The dimeric helical bundle chorismate mutase
lished using green fluorescent protein as a reporter (Supplementary
from Escherichia coli (EcCM) was successfully converted into a func-
Fig. 1 and Supplementary Methods online). The presence of the
tional hexamer (hEcCM) by inserting a five-amino-acid hinge loop into
degradation tag in plasmid pKTS also increased the dynamic range of
the middle of the long H1 helix spanning the parent dimerbut the
the chorismate mutase selection system, as demonstrated by
topological change was accompanied by a 2- to 3-order-of-magnitudedecrease in activity (Table 1). Activity was partially recovered whenhEcCM was subjected to two rounds of random mutagenesis and
Table 1 Catalytic parameters of the evolved hinge-loop variants
selection in a chorismate mutase–deficient E. coli strain (KA12/pKIMP-UAFig. 1a)Nevertheless, the best variant, tEcCM,
which contained three mutations and possessed a trimeric quaternary
structure, still had a 14-fold lower kcat value than the parent EcCM
dimer (Table 1), and further improvements were not possible because
tEcCM already conferred wild-type levels of growth to its host
In theory, selection pressure in a complementation assay can be
aThe sequences of the variants are provided in Supplementary Figure 4 online. bRef. 4,
increased by decreasing the intracellular catalyst concentration.
pH 6.5. The catalytic parameters of EcCM are similar at neutral and acidic pH. cThis work.
This might be accomplished by switching to a weakly active promoter,
Protein production and characterization is described in Supplementary Methods.
Kinetic measurements were performed at 20 1C in PBS (10 mM Na
low gene dose or inefficient ribosomal binding sites for catalyst
KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 0.1 mg/ml bovine
production. Such strategies have been profitably exploited for the
Laboratory of Organic Chemistry, ETH Zurich, Ho¨nggerberg HCI F 339, CH-8093 Zurich, Switzerland. Correspondence should be addressed to D.H.
Received 21 March; accepted 24 August; published online 16 September 2007;
Figure 1 Selection plasmid design. (a) The catalyst gene (cm) onplasmid pKECMBis constitutively expressed under control of the bla
promoter. E. coli strain KA12 is deficient in chorismate mutase activity
and also requires plasmid pKIMP-UAUC, which encodes two prephenate-
processing enzymes, for growth on minimal medium(b) Selection
plasmid pKT provides graded and homogeneous transcriptional control
of catalyst production from promoter PtetA. The tetR gene and its promoter
region are located upstream of cm, so that the TetR repressor simultaneouslyregulates transcription of catalyst and TetR repressor geneThe
tetracycline-resistance determinant of the Tn10 transposon (tetA) isintegrated in the KA12 chromosome. (c) Selection plasmid pKTS permits
graded transcriptional control and limited enzyme half-life. The ssrA
sequence is incorporated as a downstream genetic fusion to the catalyst
gene. The resulting enzyme carries the degradation tag at its C terminus,
and is directed to the ClpXP protease, where it is degraded. Plasmid
construction details are provided in Supplementary Methods online.
(400 ng/ml tetracycline), an H66R mutation occurred frequently
and was further enriched at a complementation frequency of 2 ± 1%
(200 ng/ml tetracycline). An S42L mutation was also enriched under
the latter conditions, whereas the R44C mutation, which was prevalent
before selection, occurred less frequently, presumably because itprovides no catalytic benefit. As selection pressure further increased(r100 ng/ml tetracycline), the complementation frequency dropped
to o0.1% and only false positives were observed. The latter had lost
complete suppression of cell growth in the absence of inducer for all
the degradation tag mainly through frameshift mutations, and were
tested chorismate mutase variants (Table 2). Restoration of growth
therefore presumably produced at elevated concentrations. The fact
upon addition of specific tetracycline concentrations on solid media
that the most stringent conditions only yielded false positives illus-
roughly correlated with specific activity, distinguishing the most
trates the importance of fine-tuning the selection pressure in these
weakly active variant, hEcCM, from the more active tEcCM and
experiments to maximize the yield of highly active variants.
EcCM variants. The correlation between growth rate, tetracycline
The genes of six clones selected at a complementation frequency of
concentration and specific activity was even more apparent in liquid
2 ± 1% were retransformed, and the transformants grew faster than six
culture (Fig. 2a). At the highest tetracycline concentrations tested,
out of seven variants selected at a complementation frequency of 14 ±
wild-type growth rates were achieved with the tEcCM and EcCM
5% (Supplementary Fig. 3 online). For six fast-growing variants, the
variants, but not with the weakly active hEcCM. Moreover, at
degradation tag was replaced with a (His)6 tag for protein purification,
intermediate tetracycline concentrations, cells harboring tEcCM
and the enzymatic activity of the four variants that could be produced
grew more slowly than those with wild-type EcCM. The ability to
in soluble form was determined in vitro. They showed uniformly high
control selection stringency simply by adjusting tetracycline concen-
Vmax values comparable to EcCM (Supplementary Table 1 online).
tration raises the possibility of evolving topologically novel catalysts
The highest catalytic efficiency was exhibited by variant EcCM-200/4,
which contained four mutations relative to hEcCM (A9V, S42L, H66R,
To test the utility of this system in a directed evolution experiment,
T87I; see also Supplementary Fig. 4 online). It eluted from a gel
we inserted library fragments that encode the first 93 residues of
filtration column as a trimer and catalyzed the rearrangement of
hEcCM, diversified by error-prone PCR and DNA shuffliinto the
chorismate to prephenate with a kcat of 12 s–1 and a kcat/Km of 45,000
pKTS acceptor vector in place of a stuffer fragment, in-frame with the
M–1s–1 (Table 1). The turnover number, which represents a 75-fold
last seven residues of hEcCM fused to the SsrA tag. After transforma-
improvement over hEcCM and a tenfold improvement relative to the
tion of the KA12/pKIMP-UAUC selection strain (1.5 Â
best previously characterized variant tEcCM, is similar to that of
mants), library clones were picked randomly and sequenced to check
wild-type EcCM (Table 1). This result is notable as high kcat values are
library quality. With the exception of the R44C substitution, whichoccurred in 40% of the sequences because of an apparent DNA
Table 2 Benchmark complementation assays with chorismate
shuffling artifact, mutations were evenly distributed over the entire
hEcCM gene. Aliquots of the library were plated onto selective M9c
plates containing varying amounts of tetracycline to identify activecatalysts based on their ability to complement the chorismate mutase
deficiency. The number of complementing clones decreased withdecreasing tetracycline concentration (Fig. 2b), consistent with the
hypothesis that reducing intracellular enzyme concentration increases
Sequence analysis of 96 clones revealed a mutational bias in active
variants that correlates roughly with selection stringency. The
+, cell growth; 0, no cell growth. Streak-outs of KA12/pKIMP-UAUC cells containing the
emergence and disappearance of specific mutations upon increasing
indicated selection plasmids were evaluated after 2 d of incubation at 30 1C on M9cmedium plates[tc] is the tetracycline concentration in M9c medium. pKT places the
selection pressure is illustrated in Figure 2c (see also Supplementary
gene under control of the tetracycline-inducible Ptet system, pKTS additionally encodes
Fig. 2 online). At a complementation frequency of 14 ± 5%
a degradation tag fused to the catalyst.
Figure 2 Tetracycline-dependent growth in selective M9c medium and influence of tetracycline concentration on the selection process. (a) KA12/pKIMP-
UAUC cells were transformed with the pKTS selection plasmid encoding wild-type EcCM, tEcCM or hEcCM. Growth curves were determined for each
transformant. Error bars indicate the s.d. of the curve fit in each growth experiment. (b) Complementation frequency among gene library members onselective M9c plates as a function of tetracycline concentration. (c) Mutation bias as a function of complementation frequency, determined using 24sequences originating from three independent selection experiments for each selection regime. The occurrence of false positives lacking the degradation tagand the relatively frequent mutations H66R, S42L and R44C is plotted for different selection regimes. Clones grown on nonselective rich medium plates(100% complementation) were examined to assess library size, quality and sequence diversity before selection. See Supplementary Figure 2 online foralignments of all 96 sequences used for this analysis and the Supplementary Methods for detailed experimental protocols for the liquid growth tests, library
construction and selection experiments.
important for industrial biocatalysis, where high conversion of sub-
Reprints and permissions information is available online at
In conclusion, pairing a tunable promoter with a degradation tag
can provide very low but adjustable catalyst concentrations within
cells. By providing systematic control over selection stringency, this
strategy facilitates the evolution of substantially more active enzymes
1. Taylor, S.V., Kast, P. & Hilvert, D. Angew. Chem. Int. Ed. Engl. 40, 3310–3335
than is possible with systems reliant on weak constitutive gene
2. MacBeath, G., Kast, P. & Hilvert, D. Protein Sci. 7, 325–335 (1998).
expression. Extension of this approach for regulating stringency
3. Kast, P., Asif-Ullah, M., Jiang, N. & Hilvert, D. Proc. Natl. Acad. Sci. USA 93,
should benefit any cell-based selectior screeninsystem in
4. Vamvaca, K., Butz, M., Walter, K.U., Taylor, S.V. & Hilvert, D. Protein Sci. 14,
which the setting of a threshold for minimum activity allows the best
variants to be distinguished from less interesting ones.
5. Yano, T., Oue, S. & Kagamiyama, H. Proc. Natl. Acad. Sci. USA 95, 5511–5515
6. Khlebnikov, A., Risa, O., Skaug, T., Carrier, T.A. & Keasling, J.D. J. Bacteriol. 182,
Note: Supplementary information is available on the website.
7. Hansen, L.H., Ferrari, B., Sorensen, A.H., Veal, D. & Sorensen, S.J. Appl. Environ.
This work was generously supported by the Schweizerischer Nationalfonds and
8. Lutz, R. & Bujard, H. Nucleic Acids Res. 25, 1203–1210 (1997).
9. Karzai, A.W., Roche, E.D. & Sauer, R.T. Nat. Struct. Biol. 7, 449–455 (2000).
10. Burton, S.G., Cowan, D.A. & Woodley, J.M. Nat. Biotechnol. 20, 37–45 (2002).
11. Turner, N.J. in Enzyme Assays (ed. J.L. Reymond) 139–161, (Wiley-VCH, Weinheim,
M.N., P.K. and D.H. designed research; M.N., M.B. and C.H. performed the
12. Jestin, J.L. & Kaminski, P.A. J. Biotechnol. 113, 85–103 (2004).
experiments; M.N., M.B., P.K. and D.H. analyzed data; M.N., P.K. and D.H.
13. Hillen, W. & Berens, C. Annu. Rev. Microbiol. 48, 345–369 (1994).
14. Gamper, M., Hilvert, D. & Kast, P. Biochemistry 39, 14087–14094 (2000).
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