Figure 6.

Recombinant suSLBP does not bind the stem–loop. (A) Structure of the chimeric clones, with portions of the suSLBP RBD interchanged with the human RBD. (B) The suSLBP, Xenopus SLBP1 and Drosophila SLBP were expressed in a reticulocyte lysate. Aliquots of the lysate were then tested for binding to the stem–loop in a mobility shift assay. Lane 1 is the probe, lane 2 is the suSLBP, lane 3 is xSLBP1 and lane 4 is dSLBP. There is a small amount of rabbit SLBP in the lysate (35), indicated by rSLBP. (C) The hSLBP (lane 2), a mutant of hSLBP (Y24S,Y27S; lane 3) which does not bind the stem–loop (40), and the indicated hybrid human–sea urchin SLBPs (lanes 4–7) were expressed in the reticulocyte lysate in the presence of [³⁵S]methionine (top). The lysates were incubated with the radiolabeled stem–loop and the complexes formed were resolved by native gel electrophoresis (bottom). (D) An independent preparation of the indicated hybrid SLBPs (lanes 2–5) was translated in the reticulocyte lysate in the presence of [³⁵S]methionine, and the proteins detected by autoradiography. (E) The lysates from (D) (lanes 2–5), together with a lysate expressing xSLBP1 (lanes 1 and 6), were incubated with the radiolabeled stem–loop and the complexes formed were resolved by native gel electrophoresis.