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101.
How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution. 相似文献
102.
Leuko S Goh F Allen MA Burns BP Walter MR Neilan BA 《Extremophiles : life under extreme conditions》2007,11(1):203-210
Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded
by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent
studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial
diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been
dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns
were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known
halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel
intergenic spacer profiles. ARISA–PCR, performed directly on extracted DNA from different sample sites, provided significant
insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence
analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in
this environment. 相似文献
103.
104.
We have recently reported biodegradable cationic micelles self-assembled from an amphiphilic copolymer, poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl)methyl bis(ethylene)ammonium bromide]sebacate} (P(MDS-co-CES)), which were utilized to deliver a drug and nucleic acid simultaneously, and a synergistic effect was achieved. In this paper, synthesis and characterization of the polymer is presented in details, focusing on micelle formation and DNA binding under various conditions, cytotoxicity, in-vitro degradation, and gene transfection in various cell lines. The polymer was degradable and formed micelles at very low concentrations even in an environment with high salt concentration. These micelles fabricated at pH 4.6 had an average size of less than 82 nm and zeta potential of up to 84 +/- 5 mV, displaying strong DNA binding ability. They induced high gene expression efficiency in various cell lines, which was significantly greater than poly(ethylenimine) (PEI) especially in 4T1 mouse and MDA-MB-231 human breast cancer cell lines, but they were less cytotoxic. These cationic micelles may provide a promising nonviral vector for gene delivery. 相似文献
105.
106.
107.
108.
Loll B Rückert C Hee CS Saenger W Uchanska-Ziegler B Ziegler A 《Protein science : a publication of the Protein Society》2011,20(2):278-290
The human major histocompatibility complex class I antigen HLA‐B*2705 binds several sequence‐related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross‐reactivity of cytotoxic T cells (CTL) against these HLA‐B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging‐in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross‐reactivity of these CTL with a peptide derived from voltage‐dependent calcium channel α1 subunit (pCAC, SRRWRRWNR), in which the C‐terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA‐B*2705:pCAC complex by X‐ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence‐related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F‐pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA‐B*2705 heavy chain near the N‐terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg‐Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR‐directed, HLA‐B*2705‐restricted CTL to cross‐react with HLA‐B*2705:pCAC complexes. 相似文献
109.
MacKenzie AR Langford B Pugh TA Robinson N Misztal PK Heard DE Lee JD Lewis AC Jones CE Hopkins JR Phillips G Monks PS Karunaharan A Hornsby KE Nicolas-Perea V Coe H Gabey AM Gallagher MW Whalley LK Edwards PM Evans MJ Stone D Ingham T Commane R Furneaux KL McQuaid JB Nemitz E Seng YK Fowler D Pyle JA Hewitt CN 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2011,366(1582):3177-3195
We report measurements of atmospheric composition over a tropical rainforest and over a nearby oil palm plantation in Sabah, Borneo. The primary vegetation in each of the two landscapes emits very different amounts and kinds of volatile organic compounds (VOCs), resulting in distinctive VOC fingerprints in the atmospheric boundary layer for both landscapes. VOCs over the Borneo rainforest are dominated by isoprene and its oxidation products, with a significant additional contribution from monoterpenes. Rather than consuming the main atmospheric oxidant, OH, these high concentrations of VOCs appear to maintain OH, as has been observed previously over Amazonia. The boundary-layer characteristics and mixing ratios of VOCs observed over the Borneo rainforest are different to those measured previously over Amazonia. Compared with the Bornean rainforest, air over the oil palm plantation contains much more isoprene, monoterpenes are relatively less important, and the flower scent, estragole, is prominent. Concentrations of nitrogen oxides are greater above the agro-industrial oil palm landscape than over the rainforest, and this leads to changes in some secondary pollutant mixing ratios (but not, currently, differences in ozone). Secondary organic aerosol over both landscapes shows a significant contribution from isoprene. Primary biological aerosol dominates the super-micrometre aerosol over the rainforest and is likely to be sensitive to land-use change, since the fungal source of the bioaerosol is closely linked to above-ground biodiversity. 相似文献
110.
Olivier Monteuuis Doreen K. S. Goh Charles Garcia David Alloysius Jikos Gidiman Roberto Bacilieri Gilles Chaix 《Tree Genetics & Genomes》2011,7(6):1263-1275
Forty-two different genetic origins of teak (Tectona grandis) comprising 26 open-pollinated families from a clonal seed orchard (CSO) were planted in a replicated trial under 2,500 mm
of annual rainfall and no distinct dry season, in 1997, in Sabah, East Malaysia. The trees were measured or scored for various
traits at 13, 35, 49, 61, 72, 85, 96, and 106 months after planting. Mortality rate, height (H), diameter at breast height
(DBH), volume (V), and fork height (FH) varied strongly among populations and origins. The best population means after 106 months for growth
H (21.1 m), DBH (21.1 cm), and V (278 dm3) were for the CSO families. Narrow sense heritabilities for the CSO families increased gradually with age but remained lower
after 106 months for DBH (h
2 = 0.24) and V (h
2 = 0.34) than for H (h
2 = 0.51) and FH (h
2 = 0.56). Overall, the CSO families were also straighter, less forked, and grew more vertically than the native provenance
and seed-derived sources. Such differences did not exist for flowering ability, and at 106 months, the great majority of the
trees of the various origins had not yet entered the flowering stage. Overall, at 106 months, the phenotypic correlations
between the various quantitative and qualitative traits were weak, except between straightness and bending with values higher
than 0.50. These findings confirm the usefulness of CSO for teak improvement and demonstrate the beneficial influence of wet
tropical conditions on traits of major economical importance for this species. 相似文献