耿信笃
长期从事现代分离科学基础理论、生物大分子的分离纯化、高新技术及微量分析研究。
个性化签名
- 姓名:耿信笃
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学术头衔:
博士生导师
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学科领域:
分析化学
- 研究兴趣:长期从事现代分离科学基础理论、生物大分子的分离纯化、高新技术及微量分析研究。
耿信笃,男,汉族,教授/博导, 1941年4月出生于陕西省山阳县,1960年毕业于西北大学化学系分析化学专业并留校任助教。1979年晋升为讲师,1986年破格晋升为教授,1990年成为国务院学位委员会批准的第四批博士生导师。1981年5月至1982年11月在美国明尼苏达大学做访问学者,教授会成员。1982年12月至1984年6月在美国普渡大学生化系任客座教授,1995年1月至1996年2月,任美国普渡大学化学系客座教授,2001年3月至2001年5月,任美国克累顿大学化学系客座教授。
长期从事现代分离科学基础理论、生物大分子的分离纯化、高新技术及微量分析研究。先后两次承担了国家“863高技术项目”、“七五科技攻关项目”、“国家教委优秀青年教师基金”及5次承担“国家自然科学基金”项目。作为项目负责人,1998年“重组蛋白药物示范生产线及关键设备的开发生产”项目被国家计委列为国家级重大项目,总投资2200万元。取得一系列创新性的重大成果。(1)在国际上首次提出了反相液相色谱溶质的计量保留模型,经近20年的努力使其成为一个系统地、完整的计量置换理论,还分别将沿用了近一个世纪的液—固吸附弗仑德利希和朗格缪尔经验公式从理论上推导出来,并在分离科学、物理化学、分子生物学、药学及生物工程中得到了广泛地应用。(2)1991年在国际上首次提出用液相色谱对蛋白折叠并取得突破性进展,达到国际领先水平。(3)系统研究了基因工程产品分离、纯化和折叠的新型介质、新技术和新工艺。研制成功的“变性蛋白复性及同时纯化装置”经专家鉴定达到“国际首创”和“国际领先”水平。(4)首次提出了现代分离科学理论框架,专著《现代分离科学导引》一书被教育部推荐为全国研究生教材。2004年及2006年由科学出版社分别出版了“计量置换理论及应用”和“蛋白折叠液相色谱法”两本专著。在《中国科学》、“J. Chromatography”、“化学学报”等杂志上发表论文240余篇,鉴定成果4项,技术报告6份,发明专利4项。研究成果先后获得全国科学大会奖(1978),国家发明三等奖(1984),国家教委科技进步一等奖(1988),陕西省政府成果一等奖四次(1979、1992、1998、2006),美国匹兹堡第十届国际发明与新技术博览会金奖(1994)。其成果已被国家教委列入“中国高等学校重大科技成果与研究进展选编”中。
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【期刊论文】RETENTION MODEL FOR PROTEINS IN R CHROMATOGRAPHY
耿信笃, XINDU GENG* and FRED E. REGNIER*
,-0001,():
-1年11月30日
This paper presents a retention model for proteins on an reversed-phase chro-matography support in which retention is a function of the number (Z) of solven molecules required to displace the solutte from the surface. An equation is dervied that relates the capacity factor of a protein to the displacing agent concentration and the stoichimonetry of solvent-solute displacement. Experimental tests of the model indicate that each protein has a unique Z value and that Z is directly proportional to the molecular weight of a series of protins when 60%formic acid is used as the mobile phase additive. This relationship is polypeptide solutes and the support. De-sorption curves for proteins also become more convex with incerasingly molecular weight, as predicted by the retention model. In the solvent series of methanol, etha-nol, propanol, the Z number decreases form the C1 to C3 alcohol. The Z number for any particular solvent is also related to other mobile phase additives, such as acids, and the concentration of additives.
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耿信笃, GENG, Xin Du* REGNIER, Fred E
CHINESE JOURNAL OF CHEMISTRY 2003, 21, 181-188,-0001,():
-1年11月30日
With four kinds of mobile phases, methanol-water, ethanol-water, 2-propanol and acetoritrle water (all cortairing 0.1% trilfu roacetic acid), the displacemetn between soltue and solvent in RPLC wsa proved to be universal in frontal analysis (FA), Based on the measured Z value in usual RPLC to be a corstant and the quarntitative determinnation of methanol increment in mobile phase in FA. the stoichiometric displacement (SD) betwwen insulin and methanol was directly proved by the experment. The SD was also proved to ocur only on about the bonded phase layer (BPL) without any dynamic problem of mass transfer, while in FA, the SD firstly occurs on the sufaace of the BPL and then graduallly sinks into the deeper sites comparied with a dynamic problem. Although the displaced solvent by the same solute is less in the formet case, the SD in independent, of how deep of the splute enters the BPF. In addition, the numbers of the adsorbed layer on the adsorbent sur-face, but also on the exent of the complete removal of the displace-able solvent in the BPL. The physical funda mental of the SD and the methodology for investigation were also discussed.
liquid-soild system,, reversed phase liquil chromatogra-phy,, retenion mechanism,, stoichiometric displacement,, adsorption mechanism,, insulin,, frontal analysis
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耿信笃, 边六交
中国科学,1991,(9):1~8,-0001,():
-1年11月30日
本文全面地考虑了液相色谱体系中溶质和溶剂,溶质和固定相,溶荆和固定扫分子之问的各种相互怍车甸不同种类溶剂分子在固定相表面上的竞争吸咐,提出了一个具有四组参数的溶质在液相皂谱中的计量置换深留模型以文献及实验数据对比模型进行了验证结果表明,对不同的流动相和不同的固定相体系,在多组分全浓度范围内,此模型都能良好地描述实验事实。
高效液相色涪保留模型计量置换,, 多组分体系全浓度范围
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【期刊论文】Study of the retention mechanism of proteins in hydrophobic interaction chromatography
耿信笃, XINDU GENG*, LIAN GUO and JIANHUA CHANG
,-0001,():
-1年11月30日
A stoichiometric displacement retention model for proteins based on hadro-phobic interaction chromatography (HIC) is presenter. Several methods were used to demonstrate that water is the displacing agent in this process. Salt not only affects the molar concentration of water of water, but also changes the conformation of proteins, hydro-phobic interaction forces and the number of awter moiecules in a series of hydrated protein molecules. An equation that relates the capacity foactor of proteins, K', to the water concentration and the stoichiometric displacement factor, Z (the number of water molecules required to displace a protein from ligands) was derived. The in-tercept of this equation, log I, contains a number of constants a number of constants tha relate to the affinity of protein to the ligands. There is a good linear relationship betwwen log k's and log [H2O] under different chromatographic conditions. Although Z and log I varied with pH, slope j 1.74, the logarithm of hte molar concentration of pure water. Hydrophobic interactions dominate the retention of proteins in HIC at high salt concentrations.
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耿信笃, XINDU GENG* AND FRED E. REGNIER*
,-0001,():
-1年11月30日
This paper proposes a retention model which predicts that the displacement of non-polar solutes from a revesed-phase chromatographic column is accompanied by the adsortion of a stoichiometric number (Z) of solvent molecules. The number contact surface areas. Incerasing solute contact surface area would increase Z whereas increasing solvent contact surface area presented are consistent with this model. Further predictions of the model are that (1) plots of log k' versus the inverse log of solvent concentration will be non-linear at solvent concentrations where the surface of a reversed-phase support is not fully solvated, and (2) only a portion of a reversed-phase support. Non-linearity in plost of log k' versus the inverse log of solvent concentration was in fact observed at solvent concentrations where solvation of the reversed-phase support is incomplete.
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耿信笃, Bolin Gong, Lili Wang, Chaozhan Wang, Xindu Geng
38B. Gong et al./Journal of Chromatography A, 1022 (2004) 33-39,-0001,():
-1年11月30日
The monodisperse, poly (glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0ml were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon (rhIFN-) in the extract solution containing 7.0mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN was found more than 95% and 1.3
Stationary phases,, LC, Poly (, glycidylmethacrylate-co-ethylenedimethacrylate), , Interferon, Proteins
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耿信笃, 时亚丽
中国科学,1998,32(6):1~9,-0001,():
-1年11月30日
本支依据溶液中溶质溶剜和吸附剂问相互作用的五个热力学平街,推导出了一个适用于多种吸附类型的吸附剂、溶质和溶剂的计量置换吸附模型。其核心是:溶质分子被吸附剂吸附时必然要释放出一定计量曲溶剂分子。用文献中已发表的原始数学对该甓型进行了挂验,并用奉模型的线性参数值对液一固厦咐现象进行了定量曲阐述。在Langmuir模型做了多方面比较后,发现以本模型为优。
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耿信笃, **白泉
中国科学,5002,32(5):1~13,-0001,():
-1年11月30日
变性蛋白表面的疏水氯基酸残基有与疏水色谱固定相(STHIC)颗粒相互作用的倾向,两者之间的疏水相互怍用能够抑制变性蛋白分子间的相互聚集。同时疏水色谱固定相还能在分子水平上给变性蛋白分子提供足够高的能量,使其瞬时脱水并折叠成其天然构象或不同的折叠中间体。变性蛋白在疏水界面上的折叠不仅取决于其氨基酸之间的特异性相互作用及疏水色谱固定相的结构,而且还取决于固定相和流动相之间的协同作用。同时,还提出了高效疏水相互色谱(IIPHIC)进行蛋白折叠的机理及其进行蛋白折叠时能实现质量控制的原理。在适当的色谱条件下,HPHIC可使几种变性蛋白一步实现复性及同时纯化。此外,还设计制造出了直径比柱长大得多的实验室型和制备型“变性蛋白复性及同时纯化装置,LISRPP”,该“装置”具有完全除去变性剂、使蛋白质复性,与杂蛋白分离及易于回收变性剂的“一石四鸟“功能。该“装置”对变性蛋白的复性和纯化效率与通常使用的长柱相当。在制备规模情况下,该“装置”可以在低压梯度条件下简便、快速、而经济地应用于重组蛋白药物的制备。文中以重组人干扰素。Y为例。说明了制备型“装置”在其复性及同时纯化生产工艺中的应用。
蛋白折叠, 质量控制, 疏水相互作用色谱, 纯化, 生物工程, 干扰素-γ
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耿信笃, Xindu Geng a*, Quan Bai a, Yangjun Zhang a, Xiang Li a, Dan Wu b
X. Geng et al./Journal of Biotechnology 113 (2004) 137-149,-0001,():
-1年11月30日
A new technology for renaturation with simultaneous purification of the recombinant human interferon- (rhIFN-) in downstream of biotechnology is presented. The strategies to develop the new technology in industry scale were suggested. Based on chemical equilibrium and molecular interactions, the principle of rhIFN-refolding by HPHIC was described. The kind of stationary and mobile phases were evaluated and found the former to contribute to the rhIFN-refolding more than the latter. The extract containing the rhIFN-in gram scale in 7.0mol L−1 guanidine hydrochloride solution of 700mL was directly pumped into a unit of simultaneous renaturation and purification of proteins (USRPP, 10×300 mmid.) packed by small particle packings of hydrophobic interaction chromatography and a satisfactory recovery of bioactivity and mass of the rhIFN-was obtained. With flow rate 100mLmin−1 and a gradient elution by only one step in 4h, the purity and specific bioactivity approach to 95% and 8.7×107 IU−1mg, respectively. To evaluate the goodness of the presented new technology in this study, a usual method with the renaturation by dilution method firstly and then purification with a series of LC in literature was employed to compare with each other. The obtained result in terms of purity, recoveries of mass and bioactivity, cost time as well as expenses, the former is much better than the latter. Comparing the total bioactivity of rhIFN-in the extract before to that after the renaturation by the USRPP, the total bioactivity of rhIFN-increased 62-fold.
Biotechnology, Human interferon, Protein refolding, Purification technology, High-performance hydrophobic interaction chromatography, Unit of simultaneous renaturation and purification of proteins (, USRPP),
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【期刊论文】High performance hydrophobic interaction chromatography'as a tool for protein refolding
耿信笃, Xindu Geng* and Xiaoqing Chang
,-0001,():
-1年11月30日
A method for the refolding or previously unfolded proteins with a concentrated solution of denaturing agent is presented, involving the use of high-performance hydrophobic interaction chromatography (H PHIC) to separate the denaturing agent completelyt from unfolded protein and to provide a suitable environment for its refolding The retention, peak shape and peak height in HPHIC and size-exclusion chromatography. UV spectra circular dichroic spectra and bioactivity were used to, test the possiblea and completeness of the protein refolding The proposed melhod permits the extracted solution from excherichia coli eells to be injected directly into to HPHIC column and, at the same time, the reflding and purification of the proteins to be effected the renaturation and purification of recombinant human interferon from Ecoli cells is one example of the application of the method in biotechnology
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