Excellence Cluster Rebirth, Institute of Technical Chemistry-Life Science, Leibniz University of Hannover, Callinstr.5, 30167 Hannover, Germany.
J Biotechnol. 2011 Mar 10;152(1-2):1-8. doi: 10.1016/j.jbiotec.2011.01.012. Epub 2011 Jan 22.
Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.
干细胞因子（SCF）又称为 c-kit 配体，在精子发生、黑色素生成和造血早期阶段发挥重要作用。对于后者，SCF 对于造血干细胞和祖细胞的生长和扩增是必不可少的。本文描述了从大肠杆菌中生产可溶性硫氧还蛋白融合蛋白形式的重组鼠 SCF。通常在大肠杆菌中表达 SCF 时观察到不可溶和无活性的包涵体的形成几乎完全被阻止。基于膜吸附剂技术进行纯化后，融合蛋白随后用 TEV 蛋白酶切割以释放成熟的 mSCF。经过透析和最后的纯化步骤，目标蛋白以高纯度分离。通过应用人巨核细胞系的不同测试（MTT 类似物测定、长期增殖测定）证明了 mSCF 的生物活性。此外，还测试了未切割的融合蛋白的生物活性。我们观察到了显著的活性，尽管它低于纯化的 mSCF 所显示的活性。总之，通过避免包涵体的形成，我们提出了一种用于生产 mSCF 的有效方法，mSCF 是最重要的干细胞细胞因子之一。
