Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag.

The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.

Fully automated molecular mechanics based induced fit protein-ligand docking method.

We describe a method for docking a ligand into a protein receptor while allowing flexibility of the protein binding site. The method employs a multistep procedure that begins with the generation of protein and ligand conformations. An initial placement of the ligand is then performed by computing binding site hotspots. This initial placement is followed by a protein side-chain refinement stage that models protein flexibility. The final step of the process is an energy minimization of the ligand pose in the presence of the rigid receptor. Thus the algorithm models flexibility of the protein at two stages, before and after ligand placement. We validated this method by performing docking and cross docking studies of eight protein systems for which crystal structures were available for at least two bound ligands. The resulting rmsd values of the 21 docked protein-ligand complexes showed values of 2 A or less for all but one of the systems examined. The method has two critical benefits for high throughput virtual screening studies. First, no user intervention is required in the docking once the initial binding site selection has been made in the protein. Second, the initial protein conformation generation needs to be performed only once for a given binding region. Also, the method may be customized in various ways depending on the particular scenario in which dockings are being performed. Each of the individual steps of the method is fully independent making it straightforward to explore different variants of the high level workflow to further improve accuracy and performance.

Entropic interaction chromatography: separating proteins on the basis of size using end-grafted polymer brushes.

Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (DeltaSbrush(c)) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that DeltaSbrush(c) is a strong function of both the size (MWp) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of DeltaSbrush(c) on MWp provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a wide-pore (1,000-A pores, 64-microm diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K(P)) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of K(P) values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MWp range is achieved at low to moderate grafting densities and intermediate chain lengths.