Controlling the Aggregation of Conjugates of Streptavidin with Smart Block Copolymers Prepared via the RAFT Copolymerization Technique Samarth Kulkarni, † Christine Schilli, ‡ Boris Grin, † Axel H. E. Mu ¨ ller, ‡ Allan S. Hoffman, † and Patrick S. Stayton* ,† Department of Bioengineering, University of Washington, Seattle, Washington 98195, and Makromolekulare Chemie II, Universita ¨ t Bayreuth, D-95440 Bayreuth, Germany Received February 28, 2006; Revised Manuscript Received June 22, 2006 Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M n ) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M w /M n ) 1.09). The PNIPAAm block had an M n of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M n of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 °C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 °C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized. Introduction The ability to sequentially control biomolecular recognition and activity by two different stimuli, for example, to turn activ- ities of different enzymes on or off in sequence, could open new opportunities or improve existing applications in the bio- processing and diagnostic fields. Current methods for the control of protein and enzyme activities are still largely based on nonspecific and relatively large changes in solution conditions such as temperature and pH. We have developed a different approach to the control of biomolecular recognition that is based on coupling the stimulated collapse (dehydration) or expansion (rehydration) of “smart” polymer coils with recognition events such as protein-ligand or enzyme-substrate binding reactions. The smart polymers serve as both antennae and actuators, to sense signals and respond to them, leading to control of bio- recognition events. 1 Their characteristic sharp responses in coil size and physical properties to small changes in pH, temperature, and/or UV-visible light permits rapid and precise control of molecular events. In addition to the control of bioactivities, stimuli-responsive conjugates with protein and DNA have also drawn considerable attention due to their reversible aggregation properties, which can be exploited for separations and processing applications. 2-4 Two different mechanisms have been developed for switching biomolecular activities (Figure 1). The first relies on the direct stimulation of a smart polymer chain 5 that is conjugated to a protein at a specific position relative to the protein’s active site (Figure 1A). The molecular collapse and expansion of the polymer chain as a function of temperature, pH, or specific light wavelength causes steric blocking and unblocking of the protein’s active site. To prevent the intermolecular aggregation of the protein-smart polymer conjugates, the bioconjugates are immobilized onto a solid phase support such as chromatography beads. 6-8 While this is a convenient format for some diagnostic and separations applications, it is a limitation for others. A second mechanism has been more recently developed that exploits the aggregation behavior of stimuli-responsive polymer- protein bioconjugates that are free in solution before stimulation of the smart polymer to collapse (Figure 1B). If the polymer is conjugated near the active site, then in the aggregated state, the binding site is inaccessible and the protein is turned off. 9-11 However, if the polymer is attached far away from the active site, the protein’s activity may be retained in both the free and aggregated states. * Corresponding author. E-mail: stayton@u.washington.edu. Tele- phone: 206-685-8148. Fax: 206-685-8256. Address: Department of Bio- engineering, Box 355061, University of Washington, Seattle, Washington 98195. † Department of Bioengineering, University of Washington. ‡ Makromolekulare Chemie II, Universita ¨t Bayreuth. 2736 Biomacromolecules 2006, 7, 2736-2741 10.1021/bm060186f CCC: $33.50 © 2006 American Chemical Society Published on Web 09/16/2006