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note
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Fiber gene sequences were obtained from the GenBank/EMBL databases. The accession numbers are P03275 (F2), L19194 (F4), M18369 (F5), X74660 (F8), X74659 (F9), X73487 (F12), X74658 and X74659 (F15), X14241 (F17), X94485 (F19), Y14242 (F28), X76548 (F31), X94484 (F37), P18047 (F40 long), and P14267 (F41 long). The figures were generated with RASMOL (Glaxo R&D, UK) and the F5K structure coordinates (Entrez MMDB Id 1398, Protein Data Bank accession number 1KNB).
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13044280880
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note
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Site-directed mutagenesis was performed with the Quikchange kit (Stratagene) and the baculovirus constructs pAcSC2-FSKN, pAcSG2-F9K, and pAcSG2-F41LK (6, 10). All DNA manipulations and protein expression and purification were as described (6, 10).
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17
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13044304657
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note
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Fiber mutants were expressed in insect cells. Soluble and insoluble fractions were analyzed by protein immunoblotting and detection with a polyclonal antibody to F5K (4). Assessment of solubility was made by comparing the signal in the soluble and insoluble fractions with those of wild-type F5K expression in insect cells. The F5K-specific mAbs 3D9, 3F10, and 2C9 were generated for these studies by R. Wagner (ProtoProbe Inc., Milwaukee, WI). These mAbs recognize trimeric, but not heat-denatured fiber.
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18
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13044311489
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note
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5 per well) were preincubated with the competitor protein at increasing concentrations for 1 hour at 37°C Ad.CMV-βgal (9) was added at a multiplicity of infection of 10 and incubated for 1 hour. Cells were washed twice with Dulbecco's modified Eagle's medium (DMEM) plus 5% fetal calf serum (FCS) and incubated overnight Galactosidase activity was determined with the Galacto Light assay kit (Tropix Inc.).
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19
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13044264564
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note
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Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E. Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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note
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We thank all contributing scientists and technicians.
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