Ke L D, Karaganis A G, Shain S A
Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio 78284-7836.
Protein Expr Purif. 1992 Dec;3(6):497-507. doi: 10.1016/1046-5928(92)90067-7.
We describe the preparation of vectors for T7 polymerase-driven, high-level expression of rat acidic (aFGF) or basic (bFGF) fibroblast growth factors in Escherichia coli. Following isopropyl-beta-D-thiogalactoside induction of T7 polymerase, rat aFGF or bFGF represented a major portion of the proteins synthesized by vector-transformed E. coli. Passage of cell extracts through an Amicon YM-100 membrane provided ultrafiltrates containing either aFGF or bFGF as the principal component. A single heparin-Sepharose chromatography of the ultrafiltrates yielded essentially homogenous, biologically active, recombinant rat aFGF or bFGF. By silanizing vessels and using buffers containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, we could store homogenous aFGF or bFGF preparations without significant loss during repeated freezing and thawing. Previous methods for purifying aFGF or bFGF utilized salt fractionation followed by sequential ion exchange and heparin-Sepharose chromatography. These purified aFGF or bFGF preparations routinely were stored in buffered carrier protein to minimize mitogen loss resulting from adsorption to glass or plastic surfaces. In contrast, the methods that we detail are rapid, require minimal manipulation of preparations, and permit storage of carrier-free, homogenous preparations without loss resulting from surface adsorption. The protocols can be used for preparation of homogenous aFGF or bFGF of other species and may be readily applied to the isolation and characterization of FGF-like mitogens present in cultured cell extracts, conditioned medium, or tissue preparations.
我们描述了用于在大肠杆菌中由T7聚合酶驱动的大鼠酸性（aFGF）或碱性（bFGF）成纤维细胞生长因子的高水平表达的载体的制备方法。在用异丙基-β-D-硫代半乳糖苷诱导T7聚合酶后，大鼠aFGF或bFGF占载体转化的大肠杆菌合成蛋白质的主要部分。细胞提取物通过Amicon YM - 100膜后得到的超滤液中，aFGF或bFGF作为主要成分。对超滤液进行一次肝素 - 琼脂糖层析，得到基本上均一、具有生物活性的重组大鼠aFGF或bFGF。通过对容器进行硅烷化处理并使用含有3 - [(3 - 胆酰胺丙基)二甲基铵基]-1 - 丙烷磺酸的缓冲液，我们可以储存均一的aFGF或bFGF制剂，在反复冻融过程中不会有明显损失。以前纯化aFGF或bFGF的方法是先进行盐分级，然后依次进行离子交换和肝素 - 琼脂糖层析。这些纯化的aFGF或bFGF制剂通常储存在缓冲的载体蛋白中，以尽量减少因吸附到玻璃或塑料表面而导致的促有丝分裂原损失。相比之下，我们详细介绍的方法快速，对制剂的操作要求最少，并且允许储存无载体的均一制剂，而不会因表面吸附而损失。这些方案可用于制备其他物种的均一aFGF或bFGF，并且可以很容易地应用于从培养的细胞提取物、条件培养基或组织制剂中分离和鉴定FGF样促有丝分裂原。
