Purification,physical properties and stability of intestinal trefoil factor
AIM: To obtain intestinal trefoil factor (ITF) with high purity, to explore the physical properties of purified ITF and to assay the stability in an in vitro simulated gastrointestinal environment. METHODS: ITF was isolated from the Pichia pastoris culture supernatant by ammonium sulfate precipitation, Ni-NTA affinity chromatography and ultrafiltration. Mass spectrometry a-nalysis, N-terminal amino acid sequence analysis, peptide mass fingerprint analysis and non-reducing Tricine SDS-PAGE analysis were performed. An in vitro restitution model was to be established and the stability in simulated gastrointestinal environment to be determined. RESULTS: The yield of ITF was about 50 mg/ L, with the purity after purification above 95%. The N-terminal sequence of ITF was shown to be consistent with the sequences we designed. Mass spectrometry analysis demonstrated that the molecular weight of ITF was 7879.4, approximately the same as the calculated value, 7 679. 7. Native ITF existed mainly in dimer form. ITF was robustly resistant to trypsin and pepsin, and even at a high enzyme/substrate ( w/w) ratio of 1/1, more than half of ITF existed in dimer form. Different pH values ranging from 2.0 to 12.0 had little effect on the dimer of ITF and residual dimer was greater than 90%. After water bath at 37℃ for 5 d, more than 70% of ITF was not hydrolyzed. CONCLUSION: We have produced soluble ITF with a high yield and high purity and with resistance to proteases, heal, acid and alkali. The results lay a foundation for the commercial production of ITF and provides the- oretical evidence for the oral use of ITF.
NETL
NSTL
万方数据
维普
