(405b) Stable High Affinity Streptavidin Protomers for Cell Biology Application | AIChE

(405b) Stable High Affinity Streptavidin Protomers for Cell Biology Application

Authors 

Hsu, C. K. - Presenter, University at Buffalo
Huang, H. - Presenter, University at Buffalo
Pralle, A. - Presenter, University at Buffalo


While the streptavidin-biotin interaction is the basis of
many biotechnology and medical applications, the large size of streptavidin
tetramer and the multivalency of the molecule present obstacles in some
situations. The focus of the current study is thus to engineer smaller
streptavidin variants that have sufficient stability and affinity to make the
technology accessible to other applications, including in vivo cell imaging. To
this end, we adopted a rational design approach to engineer a monomer and a
?hairline? dimer mutant with improved stability and affinity. We tested monomer
mutants containing one or more engineered disulfides to stabilize the
biotin-bound conformation. The best mutant was then engineered further by
introducing surface mutations to create an extended stabilizing salt bridge
network. The Tm = 47 °C and Kd = 60 nM of
the resulting monomer, which compare favorably with those of wt monomer (Tm
= 32 °C and Kd = 130 nM), allow the molecule to be used in a
cell biology experiment performed at 37 °C. It has long been suspected that
?hairline? interaction across the dimer-dimer interface is important for high
affinity biotin binding, but engineering such a dimer has been difficult due to
limited interface between the subunits. To engineer a stable hairline dimer, we
modeled the structure and introduced an interchain disulfide between two
designed monomer subunits. We show that the purified dimer, which elutes as a
single peak by gel filtration and has high monodispersity by dynamic light
scattering, is more stable than the substituent monomer by ~ 6 °C and has an 8
fold higher affinity compared to wt monomer, making it an ideal molecule for
applications that require a stable labeling reagent.

In addition to rational design of
streptavidin protomers, we will present results from two other parallel works for
improving the biophysical properties of streptavidin monomer. First, we have
recently demonstrated that functional streptavidin monomer can be displayed on
the yeast surface using biotin as a molecular chaperone. This allowed us to
improve the stability of rationally designed molecule by directed evolution,
which has not been possible until now due to the difficulties of folding
streptavidin in yeast. Second, we used homology modeling of streptavidin and streptavidin-like
molecules from other organisms to create a stable hybrid molecule that is
roughly 20% of the mw of wt streptavidin. Toward improving the thermodynamic
and binding properties of streptavidin monomer, we will present a comparative
analysis of designed chimeras that reveal the challenges of engineering an
optimized a monomer sequence.