Artificial Protein A

Antibodies are used extensively in diagnostics, im-munoassays, therapeutics and purifications, and can be used as probes for labelling and imaging. Monoclonal antibodies, single chain and humanized antibodies have found innumerable applications in most areas of protein chemistry, biochemistry and molecular biology. However, the availability of antibodies in their highly pure form has largely limited their range of application. Immunoglobulins are routinely purified by immobilized staphyloccal protein A (SpA) or by conventional protein purification procedures. The high clinical value of immunoglobulins and disadvantages of using potentially toxic biologics in their purification initiated a remarkable study combining the powerful tools of rational computer-aided modelling and organic synthesis to generate an artificial protein A.

The crystal structure of the Fc domain of IgG and fragment B (Fb) of SpA (Figure 3) shows involvement of a total of 32 amino acid residues over an intersurface area corresponding to 400 nm2. The primary forces holding the complex together are hydrophobic, hydrogen bonding and two salt bridges. The hydro-phobic stacking is mainly provided by residues

Figure 1 (See Colour Plate 15) Illustration of the molecular model of porcine pancreatic kallikrein with the dipeptidyl motif Arg-Phe occurring in the natural kallikrein substrate, kininogen. The model was generated by manipulating the BPTI-pancreatic kallikrein complex using Quanta 97. The residues in the BPTI inhibitor not involved in the complex were deleted, leaving residues Lys-15 and Cys-14, which were substituted with arginine and phenylalanine respectively. The dipeptide was energy-minimized and its side chains were adjusted to interact with the primary and secondary binding sites, as the Lys-Cys dipeptide does in the BPTI-porcine pancreatic kallikrein complex.

Figure 1 (See Colour Plate 15) Illustration of the molecular model of porcine pancreatic kallikrein with the dipeptidyl motif Arg-Phe occurring in the natural kallikrein substrate, kininogen. The model was generated by manipulating the BPTI-pancreatic kallikrein complex using Quanta 97. The residues in the BPTI inhibitor not involved in the complex were deleted, leaving residues Lys-15 and Cys-14, which were substituted with arginine and phenylalanine respectively. The dipeptide was energy-minimized and its side chains were adjusted to interact with the primary and secondary binding sites, as the Lys-Cys dipeptide does in the BPTI-porcine pancreatic kallikrein complex.

Figure 2 (See Colour Plate 16) Illustration of the synthetic ligand docked in the substrate-binding site of porcine pancreatic kallikrein. The ligand is an analogue of the Arg-Phe dipeptide that occurs in the natural substrate of kallikrein, kininogen, and is responsible for the enzyme-substrate complex. The benzamidine and phenethylamine moieties substituted on a triazine framework mimic the Arg-Phe dipeptide. The ligand was designed and energy-minimized in Quanta 97 and moved in the vicinity of the substrate-binding site, whence the side chains were adjusted to fit in the primary and secondary binding sites in porcine kallikrein. The aromatic ring of phenethylamine stacks in the primary binding site between Tyr-99 and Trp-215 and the benzamidine group forms several interactions with Asp-189, Ser-226, Gly-216, Pro-217 and Thr-218, forming the secondary binding site.

Figure 2 (See Colour Plate 16) Illustration of the synthetic ligand docked in the substrate-binding site of porcine pancreatic kallikrein. The ligand is an analogue of the Arg-Phe dipeptide that occurs in the natural substrate of kallikrein, kininogen, and is responsible for the enzyme-substrate complex. The benzamidine and phenethylamine moieties substituted on a triazine framework mimic the Arg-Phe dipeptide. The ligand was designed and energy-minimized in Quanta 97 and moved in the vicinity of the substrate-binding site, whence the side chains were adjusted to fit in the primary and secondary binding sites in porcine kallikrein. The aromatic ring of phenethylamine stacks in the primary binding site between Tyr-99 and Trp-215 and the benzamidine group forms several interactions with Asp-189, Ser-226, Gly-216, Pro-217 and Thr-218, forming the secondary binding site.

Phe124, Phe132, Tyr133, Leu136, Ile150 and the side chain of Lys154 on SpA and Ile 253 in IgG. Four hydrogen bonds can be identified between the s2-amido group of Gln128 (SpA) and the y-hydroxyl group of Ser254 (IgG), the ¿2-amido group of Asn130 (SpA) and the ¿1-carbonyl oxygen of Asn434 (IgG), the ^-hydroxyl of Tyr133 (SpA) and the carbonyl oxygen of Leu432 (IgG), s2-amido group of Gln311 (SpA) and the ^-carbonyl oxygen of Asn147 (IgG). Salt bridges are formed between the ¿-guanidino group of Arg146 (SpA) and the y-carboxyl group of Asp315 (IgG) and between the s-amino group of Lys154 and a sulfate ion in solution. The hydrophobic core dipeptide Phe132-Tyr133 on a helical twist of the SpA Fb region is oriented to interact with a shallow groove on IgG, comprising residues Leu251, Ile253, His310, Gln311, Glu430, Leu432, Asn434 and His435. This Phe-Tyr dipeptidyl motif is found in four highly conserved regions of SpA and each is capable of interacting with IgG from different species. If the binding pocket in IgG is made more hydrophilic by replacing the His435 with an Arg, the binding with SpA weakens, suggesting the importance of hydrophobic residues in the complex.

Li and co-workers (1998) noticed that this formed an exceptional basis for the design and synthesis of a ligand for the purification of IgG.

The biomimetic ligand (ApA) is comprised of ani-lino and tyramino substituents, mimicking the binding action of the Phe-Tyr dipeptidyl unit, spatially oriented on a triazine framework acting like the helical twist of SpA (Figure 4). A diethylamino spacer was used to immobilize the ligand on a solid support. This ligand proved to be complementary to the SpA binding site and had an affinity constant of 104 mol L_1 for IgG. ApA could purify 98% IgG from human plasma and also inhibit the binding between SpA and IgG on enzyme-linked immunosor-bent assay. Immobilized ApA showed a capacity of 20 mg IgG per gram moist weight gel. This bio-mimetic ligand could be further optimized to increase selectivity, capacity and the use of milder experimental conditions. Thus, a combinatorial library comprising 88 adsorbents was constructed for lead optimization of ligand ApA. The synthesis was inspired by the 'mix and split' procedure on a triazine scaffold and was intended to mimic the binding mechanism of ApA with improved features.

Figure 3 (See Colour Plate 17) The complex between the Fb fragment of SpA and the Fc fragment of IgG. The residues in pink represent amino acids in SpA interacting with the residues in yellow in IgG. The residues in green represent the Phe-Tyr dipeptide, key residues in holding the complex. The dotted lines represent inter-and intramolecular hydrogen bonding. The interaction involves a total of 32 amino acids spanning and intersurface area corresponding to 40 nm2. The interaction is predominantly characterized by hydrophobic interactions as well as some hydrogen bonding and two salt bridges.

Figure 3 (See Colour Plate 17) The complex between the Fb fragment of SpA and the Fc fragment of IgG. The residues in pink represent amino acids in SpA interacting with the residues in yellow in IgG. The residues in green represent the Phe-Tyr dipeptide, key residues in holding the complex. The dotted lines represent inter-and intramolecular hydrogen bonding. The interaction involves a total of 32 amino acids spanning and intersurface area corresponding to 40 nm2. The interaction is predominantly characterized by hydrophobic interactions as well as some hydrogen bonding and two salt bridges.

Combinatorial solid-phase synthesis and activity analysis suggested that a biomimetic (22/8) with 3-aminophenol and 4-amino-1-naphthol substituted on a triazinyl scaffold was able to purify IgG selectively from diluted human plasma and eluted more than 99% of bound IgG.

A similar approach was employed to design highly specific ligands for other industrially and clinically important recombinant proteins such as factor VIII and insulin. These designer ligands proved to be highly successful and were able to isolate target proteins from crude fermentation broth with high specific activities and purification factor.

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