Abstract
An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex) consisting of three ribosomal proteins (L7Ae) bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.
Highlights
Molecular complexes consisting of various, multiple biomaterials have been developed [1,2,3,4,5,6]
These complexes are new bioengineering tools for manipulating cellular functions in a highly sophisticated manner. They take advantage of multi-functional materials, such as proteins attached to another molecule that serves as a scaffold
We demonstrate a procedure for the purification of the fully loaded triangular RNP by eliminating the incomplete complexes and the starting materials by the successive use of affinity resins
Summary
Molecular complexes consisting of various, multiple biomaterials have been developed [1,2,3,4,5,6]. These complexes are new bioengineering tools for manipulating cellular functions in a highly sophisticated manner. They take advantage of multi-functional materials, such as proteins attached to another molecule that serves as a scaffold. In their most effective configurations, the scaffold must be fully loaded with the desired functional molecules. We describe a convenient and generally applicable purification method
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