ENZYMATIC RESYNTHESIS OF HUMAN IFN-GAMMA N-TERMINAL PEPTIDE

为了进一步研究N-的结构与功能的关系 末端人干扰素-γ，干扰素-γ肽(-L至18)由以下步骤合成 梅里菲尔德固相技术。重组人干扰素-γ多肽的消化 在缓冲液中合成SVS蛋白酶，在有机溶剂中合成。我们研究了 温度、浓度、酶/肽比及不同因素对酶促反应的影响 有机溶剂对消化和再合成反应的影响。我们发现 在缓冲液中对干扰素-γ多肽消化迅速。超过90%的 用1/100的酶/肽比在20min RT时消化多肽 (W/W)。 用反相高效液相色谱法对消化产物进行分析。五种小肽 用反相高效液相微凝胶系统分离和分析片段，氨基酸 序列、CD光谱和生物测定。酶促再合成进展非常顺利 高选择性。再合成的最佳条件为80%1-丙氨酸 RT时酶/肽比为1/600(w/w)。再合成反应得到了一个 在24小时内平衡大约16%。干扰素-γ的受体结合分析 多肽显示该多肽不具有受体结合活性和抗病毒作用 活动。目前正在进行研究，以制备针对该肽的抗体 协助提纯重新合成的干扰素-γ分子。

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.