Affinity purification of urinary trypsin inhibitor from human urine

Abstract

Affinity protocols for the purification of urinary trypsin inhibitor (UTI) were developed. To imitate the substrate/inhibitor-binding domain (S1 domain) of trypsin and chymotrypsin, the key amino acid residues were composed to sorbents. The sorbents were then subjected to adsorption analysis with UTI. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. The purified enzyme was subjected to SDS-PAGE, trypsin inhibitor activity and peptide map fingerprinting analysis. As calculated, the theoretical maximum adsorption (Q(max)) of two affinity sorbents entitled as S-D-G and S-S-G were 31.7 and 30.1 mg/g, respectively; the desorption constants K(d) of the two sorbents were 8.9 and 18.6 μg/mL, respectively. After the separation of UTI with S-D-G and S-S-G, reducing SDS-PAGE analysis revealed that the protein was a single polypeptide with the mass of ~66 kDa, and the purified proteins were ~95 and 97% pure, respectively; the band on gel was further confirmed with peptide map fingerprinting analysis. Protein and bioactivity recoveries were 1.3 and 75.9% with S-D-G, 1.0 and 70.2% with S-S-G, respectively.

Design and Synthesis of a Chitodisaccharide-Based Affinity Resin for Chitosanases Purification

“…Chitooligosaccharides (CHOS) are natural cationic saccharides, while the catalytic domain of chitosanases is rich in acidic amino acids [12,13]; the acid-base interactions between the two molecules can provide affinity force during affinity purification. Immobilization of a ligand onto epoxy-activated resin can be achieved via nucleophilic groups (often is primary amine) presented in the ligand [14,19,20,21,22]. Because CHOS contains an amine group at the C-2 position of the sugar ring, we thus focused our efforts on the design of CHOS-based affinity resin for purification of chitosanase.…”

“…The characterization of the interactions between chitosanases and affinity resins was carried out using equilibrium adsorption study. Scatchard analysis model was used for analysis of the desorption constant ( K d ) and the theoretical maximum adsorption capacity ( Q max ) of different affinity resins [22,29]. Various concentrations of enzymes (10 mL, 0.1–0.9 mg/mL in 20 mM Tris-HCl buffer, pH 8.0) were combined with 5 g of each type of resin to reach the adsorption equilibrium in a shaken condition.…”

Rapid and efficient one‐step purification of a serralysin family protease by using a p‐aminobenzamidine‐modified affinity medium

“…According to the Scatchard method, the data should fit the following equation (Eq. ) : …”

“…To characterize the affinity value of the enzyme and the synthesized affinity material, equilibrium adsorption characteristics were evaluated. The K d (the constant of desorption) and Q max (the theoretical maximum adsorption capacity) of the affinity medium were analyzed according to the Scatchard analysis model . Briefly, 1 mL of different concentration solutions of purified MP (ranging 0.1–0.9 mg/mL in 100 mM Tris titrated by HCl to pH 7.6) were blended with 0.5 g of pABA‐modified material and shaken for 2 h at 25°C until the solution reached the adsorption equilibrium.…”

Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

“…To characterize the interaction of MP with five different types of affinity media, an equilibrium adsorption study was performed. The constant of desorption ( K d ) and the theoretical maximum adsorption capacity ( Q max ) of these affinity media were analyzed according to the Scatchard analysis model [ 24 , 47 ]. Briefly, one milliliter of increasing concentrations of purified metalloprotease (0.1–0.9 mg/mL in 20 mM Gly-NaOH buffer, pH 8.6) was mixed with 0.5 g of each affinity medium and shaken for 2 h at 4 °C until the solution reached adsorption equilibrium.…”