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991.
992.
Cheom Gil Cheong Soo Hyun Eom Changsoo Chang Dong Hae Shin Hyun Kyu Song Kyeongsik Min Jin Ho Moon Kyeong Kyu Kim Kwang Yeon Hwang Se Won Suh 《Proteins》1995,21(2):105-117
Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P42212 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (VM) of 2.57 Å3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)8 core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)8 barrel. © 1995 Wiley-Liss, Inc. 相似文献
993.
The synthetic antibody model “M41” was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine CK and CH 1γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P212121 with unit cell dimensions a = 104.7 Å, b = 113.9 Å, c = 98.8 Å and diffracted X-rays to a nominal resolution of 2.5 Å. © 1995 Wiley-Liss, Inc. 相似文献
994.
995.
996.
Shinji Hosoi Mitsuo Satoh Hiromasa Miyaji Tatsunari Nishi Tamio Mizukami Mamoru Hasegawa Seiga Itoh Tatsuya tamaoki 《Cytotechnology》1995,19(1):1-10
Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml–1. Addition of glucose and tri-iodothyronine (T3) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml–1 day–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA
bovine serum albumin
- dhfr
dihydrofolate reductase
- HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- kb
kilobase pairs
- kDa
kilodaltons
- MTX
Methotrexate
- PBS
phosphate buffered saline
- pro-UK
pro-urokinase
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- T3
tri-iodothyronine
- Tween-PBS
phosphate buffered saline containing 0.05% Tween 80 相似文献
997.
Rasmus H. Fogh Dick Schipper Rolf Boelens Robert Kaptein 《Journal of biomolecular NMR》1995,5(3):259-270
Summary The 1H, 13C and 15N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CHn carbons, all amide (NH and NH2) nitrogens and 99.2% of the amide and CHn protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D
two-/three-/four-dimensional
- HSQC
heteronuclear single-quantum coherence
- HMQC
heteronuclear multiple-quantum coherence
- COSY
correlation spectroscopy
- TOCSY
total correlation spectroscopy
- NOE
nuclear Overhauser enhancement (connectivity)
- NOESY
2D NOE spectroscopy
Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667]. 相似文献
998.
Jo Oldknow Tanya M. Franklin Martin Trick Sharon Allard Laurian S. Robert 《Sexual plant reproduction》1995,8(4):254-255
The DNA sequence data reported have been lodged in the Genbank, EMBL and DDBJ databases under the accession numbers Z21609 and Z26914 相似文献
999.
Marianne Borloo Koen Augustyns Alexander Belyaev Ingrid de Meester Anne-Marie Lambeir Filip Goossens Willy Bollaert Padinchare Rajan Simon Scharpé Achiel Haemers 《Letters in Peptide Science》1995,2(3-4):198-202
Summary A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range. 相似文献
1000.
We have used antibodies directed against a unique portion of the Drosophila POU domain protein Cfla to localize its sites of expression in developing embryos. Cfla protein is first detected during germ band extension in the tracheal placodes and in the midline mesectoderm cells. Tracheal expression continues throughout embryonic development, especially in the main longitudinal tracheal trunks. Additional sites of high Cfla expression are in the anterior portion of the hindgut, the roof of the stomodeum, a subset of central nervous system cells, the oenocytes, and the ring gland. In addition, Cfla expression was localized in embryos mutant for several loci involved in determining fate along the midline of the CNS and the tracheal system. Cfla midline cell expression is dependent on proper single-minded gene function, and Cfla either regulates or acts in parallel to the genes pointed and rhomboid during midline CNS and tracheal development. 相似文献