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Abstract
Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.
Highlights
An ideal affinity purification system for recombinant proteins should purify the target proteins from the crude extract in one step under mild conditions with high purity and excellent recovery in a cost-effective manner
We reported the development of an engineered streptavidin designated SAVSBPM18 with these desirable properties
Out of 38 residues in the full-length streptavidin-binding peptide (SBP) tag, only 25 amino acids form a well-ordered structure that can be visualized in the streptavidin-SBP tag complex
Summary
An ideal affinity purification system for recombinant proteins should purify the target proteins from the crude extract in one step under mild conditions with high purity and excellent recovery in a cost-effective manner. Biotinylation of molecules either chemically [3] or enzymatically [4] requires extra steps which can be time consuming, labour intensive and costly To overcome this problem, several streptavidin-binding peptide tags have been developed. One of them is the 38-amino-acid streptavidin-binding peptide (SBP) tag [5] that can bind streptavidin with high affinity (Kd ,2.5 nM) without biotinylation. As streptavidin binds biotin tightly (Kd ,10214 M) [9], the streptavidin matrix essentially can be used only once The use of this purification approach can be relatively costly. To vitalize this powerful purification technology, it would be ideal to have an engineered streptavidin that can bind biotin reversibly while retaining a high SBP tag binding strength. Its application for purification of a SBP-tagged protein and a biotinylated protein was demonstrated
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