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Fig. 7. TAP purification of SRP. (A) Fractionation of RNP particles on a sucrose gradient. Whole cell extract (150 ml culture, 107 cells/ml) was prepared from induced cells carrying the SRP19TAP construct. The extract was layered on a continuous 10-30% (w/v) sucrose gradient. The gradient was fractionated as described in Fig. 5. RNA extracted from an aliquot of odd-numbered fractions was separated on a 10% polyacrylamide-7M urea gel and subjected to northern analysis with random-labeled 7SL RNA. Fraction numbers are indicated (from top to bottom). Proteins extracted from an aliquot of even numbered fractions were separated on a 12% SDS-polyacrylamide gel and subjected to western analysis with IgG antibodies. Fraction numbers are indicated (from top to bottom). S values were determined relative to 28S rRNA, 4S RNA and catalase (11S). The transcripts of 7SL RNA and the SRP19TAP protein are indicated by arrows. (B) Immunoprecipitation of the SRP by IgG. Whole cell extract was prepared from induced cells (50 ml, 107 cells/ml) carrying the SRP19TAP construct and subjected to affinity selection using IgG Sepharose. The immunoprecipitated products and 2% of total cell extract and supernatant were separated on a 12% SDS-polyacrylamide gel and subjected to western analysis with IgG antibodies. SRP19TAP is indicated by an arrow. (C) Purification of SRP proteins. Whole cell extracts (1 litre, 107 cells/ml) were prepared from induced cells carrying the SRP19TAP construct and from uninduced cells (control) and the extracts were subjected to affinity selection using IgG Sepharose. Proteins from uninduced cells are shown in lane 1, and induced cells in lane 2. Complexes selected with IgG beads were treated with TEV protease and were further purified by DEAE column (lane 3). Proteins extracted from the eluate were fractioned on a 10% SDS-polyacrylamide gel and stained with silver. SRP21 (SRP19 homolog fused to CBP), SRP19TAP (
40 kDa), SRP54, SRP64 (SRP68 homolog) and SRP75 (SRP72 homolog) are indicated by arrows. (D) Purification of SRP RNAs. As for C except that RNA was prepared from the affinity selection fraction, separated on a 10% polyacrylamide-7M urea gel and stained with silver. 7SL RNA and sRNA-76 are indicated by arrows. (E) Immunofluorescence assay (IFA) of the SRP19TAP protein. Cells carrying the SRP19TAP construct were induced for 4 hours (1) and 60 hours (2) and the tagged protein was visualized. Cells were fixed with 4% formaldehyde for 25 minutes, incubated with IgG and visualized as described in Materials and Methods. Bar, 5 µm.
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