ENZYMATIC RESYNTHESIS OF HUMAN IFN-GAMMA N-TERMINAL PEPTIDE

人干扰素-γ N 端肽的酶法再合成

• 批准号: 3811194
• 负责人: R Q HU
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3811194
• 关键词: enzyme substrate; high performance liquid chromatography; interferons; nonwater solvent; peptidases; peptide chemical synthesis; protein sequence; protein structure function; receptor binding

In order to further study relationship of structure and function of N- terminal human IFN-gamma, IFN-gamma peptide (-l to 18) was synthesized by the solid-phase technique of Merrifield. IFN-gamma peptide was digested wit SVs protease in buffer and resynthesized in organic solvent. We studied the effect of temperature, concentrations enzyme/peptide ratio and different organic solvents on reaction of digestion and resynthesis. We found digestion of IFN-gamma peptide in buffer was rapid. Greater than 90% of peptide was digested at 20 min RT using an enzyme/peptide ratio of 1/100 (w/w). The digestion products were analyzed by RP-HPLC. Five small peptides fragments were isolated and analyzed by RP-HPLC mini-gel system, amino acid sequence, CD spectra and bioassay. Enzymatic resynthesis proceeds with very high selectivity. Optimal condition for resynthesis was 80% 1-proponal enzyme/peptide ratio 1/600 (w/w) at RT. Resynthetic reaction attained an equilibrium approximately 16% in 24 hrs. Receptor binding assay of IFN-gamm peptide revealed the peptide had no receptor binding activity and antiviral activity. Studies are in progress to prepare antibodies to the peptide to assist in purifying the resynthesized IFN-gamma molecules.

为了进一步研究N-的结构与功能关系