Belew M, Zhou Y, Wang S, Nyström L E, Janson J C
Pharmacia Bioprocess Technology, Uppsala, Sweden.
J Chromatogr A. 1994 Sep 9;679(1):67-83. doi: 10.1016/0021-9673(94)80312-9.
Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), produced as inclusion bodies in genetically transformed Escherichia coli cells was purified to homogeneity by a three-step chromatographic procedure involving hydrophobic interaction, ion exchange and gel filtration. Each purification step is reproducible and well suited for process-scale operations. The purification process also leads to a significant decrease in DNA and endotoxin levels in the final product. Of the three gel media used, Phenyl Sepharose 6 FF (high sub) was most effective in reducing the DNA content (by a factor of ca. 2000) while Superdex 75 prep grade was more effective for removing endotoxins (reduction factor ca. 15). The recovery of purified rhGM-CSF was 35% by enzyme-linked immunosorbent assay and 70% by a biological assay method. The overall purification factor obtained was about 4.6, which is in the range of those reported for recombinant proteins produced in E. coli as inclusion bodies. The purified rhGM-CSF is an acidic protein (pI = 5.4) and has a specific activity of ca. 3.3 x 10(7) units/mg, which is in excellent agreement with that reported for its natural counterpart. Its monomer molecular mass of 14,605, as determined by electrospray mass spectrometry, corresponds exactly to the mass calculated from its cDNA sequence. Its amino acid composition and partial NH2-terminal sequence (up to seventeen residues) are also identical with those reported for this protein. These and other results confirm the identity of the purified rhGM-CSF with its natural counterpart. However, the results also showed that it is apparently heterogeneous from its NH2-terminal side as it is composed of three polypeptides having Met, Ala and Pro as the NH2-terminal residues in which the intact Met analogue accounts for 60% for the mixture. This heterogeneity does not seem to have any biological significance since the specific activity of the purified rhGM-CSF is identical with that of its natural counterpart.
在基因转化的大肠杆菌细胞中作为包涵体产生的重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF），通过疏水相互作用、离子交换和凝胶过滤三步色谱法纯化至均一。每个纯化步骤均可重现，且非常适合大规模生产操作。该纯化过程还导致最终产品中的DNA和内毒素水平显著降低。在所使用的三种凝胶介质中，苯基琼脂糖6 FF（高取代度）在降低DNA含量方面最有效（降低约2000倍），而超级葡聚糖75预装级在去除内毒素方面更有效（降低因子约为15）。通过酶联免疫吸附测定法，纯化的rhGM-CSF的回收率为35%，通过生物学测定法为70%。获得的总体纯化因子约为4.6，处于报道的在大肠杆菌中作为包涵体产生的重组蛋白的范围内。纯化的rhGM-CSF是一种酸性蛋白（pI = 5.4），比活性约为3.3×10⁷单位/毫克，与其天然对应物的报道值非常一致。通过电喷雾质谱法测定，其单体分子量为14,605，与根据其cDNA序列计算的质量完全一致。其氨基酸组成和部分NH₂末端序列（多达十七个残基）也与该蛋白的报道值相同。这些结果及其他结果证实了纯化的rhGM-CSF与其天然对应物的一致性。然而，结果还表明，从其NH₂末端来看它显然是异质的，因为它由三种以Met、Ala和Pro作为NH₂末端残基的多肽组成，其中完整的Met类似物占混合物的60%。这种异质性似乎没有任何生物学意义，因为纯化的rhGM-CSF 的比活性与其天然对应物相同。
