Pathak N, Hu S I, Koehn J A
Core Technologies, Arthritis Biology, Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, New Jersey, 07901, USA.
Protein Expr Purif. 1998 Nov;14(2):283-8. doi: 10.1006/prep.1998.0972.
We have cloned, expressed, and purified a recombinant C-terminal truncated form (residues Glu103-Asn274) of human collagenase-3 (MMP-13) in Escherichia coli. The molecule contains the catalytic domain of the enzyme and is expressed almost exclusively as inclusion bodies. Using a combination of rapid dilution and diafiltration, the enzyme has been successfully refolded from these inclusion bodies. The protein was purified to homogeneity using cation-exchange and size-exclusion chromatography. The purified enzyme is a monomer with a Mr of approximately 19,600 and was characterized using a variety of techniques including, SDS-PAGE, RP-HPLC, LC-MS, amino acid analysis, and dynamic light scattering. Microheterogeneity at the NH2-terminus of the refolded, purified protein disappeared after incubating for 30-60 min at 37 degreesC. The enzyme was highly active using a fluorescent peptide substrate and was found to release S-GAG from bovine nasal cartilage chips.
我们已在大肠杆菌中克隆、表达并纯化了人胶原酶-3（MMP-13）的重组C端截短形式（Glu103-Asn274残基）。该分子包含酶的催化结构域，几乎完全以包涵体形式表达。通过快速稀释和渗滤相结合的方法，该酶已成功地从这些包涵体中复性。使用阳离子交换和尺寸排阻色谱法将蛋白质纯化至均一。纯化后的酶是一种单体，Mr约为19,600，并使用多种技术进行了表征，包括SDS-PAGE、RP-HPLC、LC-MS、氨基酸分析和动态光散射。复性纯化后的蛋白质在37℃孵育30-60分钟后，其NH2末端的微不均一性消失。该酶使用荧光肽底物时具有高活性，并能从牛鼻软骨碎片中释放S-GAG。
