title: 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.
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu, P. R. China. marshalxy@gmail.com
authors: Yu
authors: Y
authors: Xin
authors: Hailin
authors: H
authors: Yang
authors: Xiaole
authors: X
authors: Xiao
authors: Ling
authors: L
authors: Zhang
authors: Yuran
authors: Y
authors: Zhang
authors: Yanjun
authors: Y
authors: Tong
authors: Yi
authors: Y
authors: Chen
authors: Wu
authors: W
authors: Wang
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000652:urine; D014462:Ultrafiltration / Q000379:methods*
chemical_list: D014361:Trypsin Inhibitors