在乙腈存在下的疏水相互作用色谱法和阴离子交换色谱法。一种人催乳素的两步纯化方法。

Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin.

作者信息

Hodgkinson S C, Lowry P J

出版信息

Biochem J. 1981 Dec 1;199(3):619-27. doi: 10.1042/bj1990619.

DOI:10.1042/bj1990619

PMID:7340823

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1163418/

Abstract

Described is a two-chromatographic-step preparative-scale technique for the purification of human prolactin from a frozen pituitary homogenate. The method utilizes hydrophobic interaction chromatography on the mildly hydrophobic adsorbent phenyl-Sepharose CL-4B and anion-exchange chromatography on DEAE-cellulose in the presence of acetonitrile. Human prolactin was solubilized at pH10.0 after a prior extraction of pituitaries at pH4.0, the acid pH being ineffective at solubilizing human prolactin but capable of solubilizing large amounts of interfering protein. An 11-fold increase in the potency of the solubilized human prolactin was achieved in this manner. Prolactin could be adsorbed to phenyl-Sepharose at low ionic strengths (I<0.01); few other proteins were adsorbed under these conditions. This is a demonstration of the hydrophobic nature of human prolactin. The amount of phenyl-Sepharose was limited to the minimum (35mg of protein/g of phenyl-Sepharose) necessary to adsorb human prolactin, further reducing the uptake of other pituitary protein. Desorption was achieved by using an acetonitrile gradient (0-30%, v/v), resulting in a purification of human prolactin of 85-fold and recovery of 78%. Acetonitrile (20%, v/v) was also included in all buffers for DEAE-cellulose chromatography, increasing the resolution and recovery of human prolactin, apparently by minimizing non-ionic interactions with the matrix. Prolactin (10mg) was recovered from 63g if pituitaries, an overall recovery of 58%. It was homogeneous by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, contained less than 0.1% somatotropin (growth hormone), on iodination demonstrated more than 95% binding to excess anti-(human prolactin) serum and could be displaced from anti-(human prolactin) serum in a manner indistinguishable from the serum of a patient with a human prolactin-secreting adenoma.

摘要

本文描述了一种从冷冻垂体匀浆中纯化人催乳素的两步制备规模色谱技术。该方法利用轻度疏水吸附剂苯基琼脂糖CL-4B进行疏水相互作用色谱，以及在乙腈存在下在DEAE-纤维素上进行阴离子交换色谱。在pH4.0预先提取垂体后，人催乳素在pH10.0下溶解，酸性pH对溶解人催乳素无效，但能够溶解大量干扰蛋白。通过这种方式，溶解的人催乳素的效价提高了11倍。催乳素可以在低离子强度（I<0.01）下吸附到苯基琼脂糖上；在这些条件下很少有其他蛋白质被吸附。这证明了人催乳素的疏水性。苯基琼脂糖的用量限制在吸附人催乳素所需的最小值（每克苯基琼脂糖35mg蛋白质），进一步减少了其他垂体蛋白的摄取。通过使用乙腈梯度（0-30%，v/v）实现解吸，从而将人催乳素纯化85倍，回收率为78%。在所有用于DEAE-纤维素色谱的缓冲液中也加入了乙腈（20%，v/v），这显然通过最小化与基质的非离子相互作用提高了解析度和人催乳素的回收率。从63g垂体中回收了10mg催乳素，总回收率为58%。通过凝胶过滤和十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示其为均一的，含有少于0.1%的生长激素，碘化后显示与过量抗（人催乳素）血清的结合率超过95%，并且可以以与分泌人催乳素腺瘤患者血清无法区分的方式从抗（人催乳素）血清中被置换出来。