Purification of streptavidin

Streptomyces avidinii can be obtained from the American Type Culture Collection (ATCC 27419) and grown in a medium according to Stapley et al. (1963) on 2% agar plates at 30 C. Spores from four plates are used to inoculate 4 1 of medium, which is then incubated for 3 days at 30 C. The medium is clarified by centrifugation (10 min, 10000 xg) and concentrated in an Amicon concentrator (PM 10 membrane) to 400 ml. To this solution at 0 C, (NH4)2S04 

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Bayer, E., Ben-Hur, H., Citlin, G., and Wilchek, M. (1986) An improved method for the single-step purification of streptavidin./. Biochem. Biophys. Meth. 13, 103-112. 

For purification, load the reaction mixture onto a 9 x 200 mm Bio-Gel P-6DG column packed in PBS (0.1% BSA may be added as a carrier to the PBS to reduce loss of streptavidin by adsorption to the column) Elute the column with PBS and collect 0.5-mL fractions The first set of radioactive fractions (as determined by counting in a y-ray counter) contains radioiodinated streptavidin, while the unreacted radioiodine elutes in the later fractions. Pool the radioiodinated streptavidin fractions. 

Figure 8.14. Affinity purification using magnetic particles purification of biomolecules using streptavidine and biotin affinity.

One of the approaches has been to genetically design fusion proteins with an affinity domain linked to the enzyme. This technology provides for one-step purification as opposed to the multistep processes required for purification of commercial enzymes. Furthermore, immobilization can be achieved at the same time, leading to a minimization of bioreactor preparation costs. Fusion proteins with streptavidin as an affinity domain have been designed (Sano and Cantor, 1991 Walsh and Swaisgood, 1994 Lee and Swaisgood, 1998). 

After purification of mRNA, it is reverse-transcribed to cDNA, which is labeled with two different fluorescent tags. Equal amounts of cDNA preparation are mixed and applied to die microarray for competitive binding. (B) Most steps for Affymetrix microarrays are similar. For labeling biotin is used and readout of expression values is based on a streptavidin tag binding to biotin. Compared to Agilent, only one sample is hybridized per array. 

Figure 12.5 illustrates schematically the triplex-mediated capture procedure for the purification of specific ds DNA as described by Ito et al. (1992). A biotinylated pyrimidine oligomer is incubated under acidic conditions (pH 4.5-5.0), since the cytosines should be protonated, with a DNA mixture containing the target. The triplex is then captured with paramagnetic beads coated with streptavidin, the beads washed several times and the retained duplex eluted. 

A. D. Keefe et al. One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expression and Purification, 23 (2001), 440-6. 

Fig. 4.3. Affinity purification of RNA associated complexes. The RNA may be biotinylated during transcription and complexes are allowed to form in solution. The complexed RNA can then be immobilised on streptavidin coated beads and purified. Associated molecules can be eluted from the RNA and analysed by the method of choice. Variations of this scheme are described in the text. In certain cases it may be favourable to form the complexes after the immobilisation step.

Rybak, ).N., Scheurer, S.B., Neri, D., and Elia, G. (2004) Purification of biotinylated proteins on streptavidin resin a protocol for quantitative elution, Proteomics 4, 2296-2299. 

Fig. 8 Overcoming the purification bottleneck via immobilization of streptavidin isoforms using biotin-sepharose [57]...

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