IFBM'S to promote attachment of target analytes
A technology of analytes and implants, applied in the direction of analyzing materials, instruments, prostheses, etc., can solve problems such as slow wound healing and improper integration of implants with surrounding tissues
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Embodiment 1
[0061] Example 1: Isolation of titanium-binding polypeptides
[0062] Ten different phage display libraries were used to screen for peptides bound to titanium beads. Titanium (Ti 6 Al 4 v) Beads to remove any surface contamination. Each titanium bead was placed in each well of a 96-well polypropylene plate (Nunc).
[0063] Nonspecific binding sites on the titanium and polypropylene surfaces were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS; Sigma Chemical Company, St. Louis, MO, Cat. No. P-3813). Plates were incubated for 1 hour at room temperature with shaking at 50 rpm. The wells were then washed 5 times with 300 μl PBS. Each library was diluted in PBS+1% BSA at 10 10 A concentration of pfu / ml was added to 250 μl. After culturing with shaking at 50 rpm for 3 hours at room temperature, use 300 μl of phosphate buffer-Tween TM 20 (PBS-T; Sigma Chemical Company, St. Louis, MO, cat. no. P-3563) was washed 3 times to remove unbound phage. ...
Embodiment 2
[0070] Example 2: Role of Cysteine Residues in Titanium-Binding Polypeptide 6007
[0071] To investigate the role of cysteine residues and disulfide structures in the binding of peptide 6007 to titanium, the cysteine residue present in the titanium-binding peptide AFF6007 was converted to a serine residue to synthesize AFF6010 (Table 2 ). The sequence of polypeptide AFF6010 (SSSDKSHKHWYSYESKYGGSGSSGK) is shown in SEQ ID NO:9, and the sequence of polypeptide AFF6007 (SSSDKCHKHWYCYESKYGGSGSSGK) is shown in SEQ ID NO:10. Peptides AFF6007 and AFF6010 were then conjugated to biotin and compared for binding to titanium beads as follows.
[0072] Titanium beads were blocked with 1% BSA in PBS for 30 min at room temperature. Stock solutions of polypeptides AFF6007 and AFF6010 were prepared by dissolving 1-2 mg of polypeptide in water. The final concentration of each polypeptide was determined using the absorbance at 280 nm and the absorbance coefficient of each polypeptide. ...
Embodiment 3
[0077] Example 3: Polypeptides that specifically bind to bone morphogenetic protein 2 ("BMP-2")
[0078] Separation and Analysis of Peptides
[0079] Ten different phage display libraries were used to screen for polypeptides that bind to BMP-2. BMP-2 (Medtronic) was biotinylated with NHS-biotin (Pierce) to prepare labeled proteins, with an average of one biotin bound per protein molecule. The protein was immobilized on streptavidin (SA)-coated plates for phage display. There is another method of displaying proteins, according to the manufacturer's instructions (Amersham-Pharmacia, Ref. No. 18-1022-29, titled "Coupling through the Primary Amine of a Ligand to NHS activated Sepharose 4 Fast Flow", pages 105-108) Using NHS-succinimide chemistry, BMP-2 was bound to Sepharose beads and the beads were used as a solid phase to separate unbound phage. After 3 rounds of selection, individual clones selected from each format were tested for their ability to bind BMP-2 on SA-coated pl...
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