Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein

Abstract

Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin-biotin for composite separation or analysis steps.

Fig. 1.

Construction of circularly permuted single-chain dimeric streptavidin. Two WT genes (a) are cut, spliced, and provided with tetrapeptide linkers (b) L and R (Left and Right) designate the N- and C-terminal halves of this construct. (c) Structural representation with segments colored in order from N to C terminus, blue, green, yellow, and brown. GGGS linkers are depicted as ball-and-stick models. Residue numbering corresponds to WT streptavidin with primes added to distinguish positions in the R domain.

Fig. 2.

Expression of SCD proteins in E. coli carrying pET22b(+) monitored on SDS/PAGE (4–20% VWR i-gel). (a) SCD, (b) C2, and (c) E2, as marked. Lane headings: M, protein marker (bands from top to bottom in kDa: 94, 67, 43, 30, 20, and 14.4); U, uninduced culture; I, induced culture; and 0, 2, hrs after start of induction. Expression was at 37°C and 1 mM IPTG from 500 ml of culture volume. Inclusion bodies from 500 μl of culture were loaded on the gel for SCD and from 250 μl for C2 and E2. Marker lanes contained 58 μg of protein for a and c and 29 μg for b.

Fig. 3.

FPLC of mutant E2 fractions. T and D refer to tetramer and dimer. (a) FPLC of crude E2. b and c show results of rechromatography of peaks without biotin from the crude fraction shown in a for E2T and E2D, respectively. The rechromatographed E2D peak from c was incubated with added biotin and rerun on the column, yielding only a tetramer peak (d).

Fig. 4.

Fluorescence intensity and polarization behavior of B4F binding to mutant E2. Quenching curves for (a) dimer and (d) tetramer fractions. Protein samples with the final monomer concentrations shown were mixed with 0.16 nM B4F in a total volume of 150 μl. Blue and red symbols correspond to duplicate samples. Competition experiments for (b) dimer and (e) tetramer monitored by polarization. Protein samples (9 nM monomeric binding sites) were first incubated with 0.16 nM B4F and then chased with different concentrations of biotin: blue, no biotin; brown, 492 μM biotin; and green, 725.2 μM biotin. Reverse-competition experiment for (c) dimer and (f) tetramer. Protein samples (9 nM monomeric binding sites) were mixed with 45 nM biotin (red points) then chased with B4F (final concentration, 0.16 nM). Blue points, no biotin. Note the truncated y axes in c and f.