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. 1998 Sep 21;188(6):1091–1103. doi: 10.1084/jem.188.6.1091

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Analysis of the expression of Hbp by Hbp-mutants. The mutants mHbp18 and mHbp1 were obtained by site-directed mutagenesis experiments with the suicide vectors pSG-Hbp2.2 and pSG-Hbp4.8, respectively. See Materials and Methods for a detailed description of the construction of these suicide vectors. Culture supernatant fractions and total lysate fractions were derived from 4.0 and 0.5 OD₆₆₀ units, respectively. The fractions were analyzed by immunoblotting with the anti-Hbp antiserum. (A) Expression of Hbp by mHbp18. Lanes 1 and 4, lysate and supernatant of the wild-type strain EB1; lanes 2 and 3, lysate and supernatant of mHbp18. (B) Expression of Hbp by mHbp1. Lanes 1 and 4, same samples as in A; lanes 2 and 3, lysate and supernatant of mHbp1. The secretory intermediates of Hbp and molecular mass markers (kD) are indicated at the right and left side of the panels, respectively. A schematic representation of the recombination events of the suicide vectors is depicted at the right side of this figure. Mutant mHbp18 was the result of a double crossover and mutant mHbp1 of a single crossover event. Symbols in this diagram are: M, MluI restriction site; P, promoter; km, kanamycin cassette.

Analysis of the expression of Hbp by Hbp-mutants. The mutants mHbp18 and mHbp1 were obtained by site-directed mutagenesis experiments with the suicide vectors pSG-Hbp2.2 and pSG-Hbp4.8, respectively. See Materials and Methods for a detailed description of the construction of these suicide vectors. Culture supernatant fractions and total lysate fractions were derived from 4.0 and 0.5 OD₆₆₀ units, respectively. The fractions were analyzed by immunoblotting with the anti-Hbp antiserum. (A) Expression of Hbp by mHbp18. Lanes 1 and 4, lysate and supernatant of the wild-type strain EB1; lanes 2 and 3, lysate and supernatant of mHbp18. (B) Expression of Hbp by mHbp1. Lanes 1 and 4, same samples as in A; lanes 2 and 3, lysate and supernatant of mHbp1. The secretory intermediates of Hbp and molecular mass markers (kD) are indicated at the right and left side of the panels, respectively. A schematic representation of the recombination events of the suicide vectors is depicted at the right side of this figure. Mutant mHbp18 was the result of a double crossover and mutant mHbp1 of a single crossover event. Symbols in this diagram are: M, MluI restriction site; P, promoter; km, kanamycin cassette.