Kumar Lokender, Colomb Warren, Czerski John, Cox Christopher R, Sarkar Susanta K
Single Molecule Biophysics Laboratory, Department of Physics, Colorado School of Mines, 1500 Illinois Street, Golden, CO 80401, USA.
Advanced Biodetection Technology Laboratory, Department of Chemistry, Colorado School of Mines, 1500 Illinois Street, Golden, CO 80401, USA.
Protein Expr Purif. 2018 Aug;148:59-67. doi: 10.1016/j.pep.2018.04.001. Epub 2018 Apr 4.
MMP1 is an essential enzyme for tissue remodeling both in normal and pathological states. We report a method of purifying activated human MMP1 in E. coli without using urea or 4-Aminophenylmercuric acetate (APMA). Instead, a non-ionic detergent, Triton X-100, was used in the lysis buffer to solubilize MMP1 followed by the protease activities of both trypsin and MMP1 to digest E. coli proteins and activate pro-MMP1. Identity of activated MMP1 was confirmed by Western blot using anti-human MMP1 antibodies, whereas the mass was determined to be 43 kD using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS). Collagen and gelatin degradation by purified MMP1 were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of degraded FITC-labeled type-1 collagen and gelatin zymogram. Broad-spectrum protease activity of purified MMP1 was also confirmed by lysis of native E. coli proteins. Inexpensive high throughput purification of recombinant human MMP1 in E. coli will enable easier MMP1 production for diverse applications.
基质金属蛋白酶1（MMP1）是一种在正常和病理状态下对组织重塑至关重要的酶。我们报道了一种在大肠杆菌中纯化活化人MMP1的方法，该方法不使用尿素或对氨基苯基汞乙酸盐（APMA）。相反，在裂解缓冲液中使用非离子去污剂 Triton X-100 来溶解 MMP1，随后利用胰蛋白酶和 MMP1 的蛋白酶活性消化大肠杆菌蛋白并激活前 MMP1。使用抗人 MMP1 抗体通过蛋白质免疫印迹法确认活化 MMP1 的身份，而使用基质辅助激光解吸电离飞行时间质谱（MALDI TOF-MS）测定其质量为 43 kD。通过对降解的异硫氰酸荧光素标记的 I 型胶原蛋白和明胶酶谱进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS PAGE），确认纯化的 MMP1 对胶原蛋白和明胶的降解作用。通过裂解天然大肠杆菌蛋白也证实了纯化的 MMP1 的广谱蛋白酶活性。在大肠杆菌中对重组人 MMP1 进行廉价的高通量纯化将使 MMP1 的生产更易于用于各种应用。
