Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease caused by degeneration of motor neurons. Not until recently ALS has been associated with specific mutations in ANXA11 gene. Annexin A11 (ANXA11) belongs to a larger annexin family of structurally related Ca2+-dependent-membrane-binding proteins. The C-terminal core contains four highly conserved homologous alpha helical repeats. Contrary, the N-terminus is highly variable, structurally disordered and the longest in the annexin family. It is important for nuclear localization and degradation of ANXA11. Recombinant ANXA11 was previously prepared by master's student Jakob Rupert, using pET28a and pET14b vectors. Due to protein degradation problems we decided to attempt production using an alternative vector system. With ligation independent cloning we incorporated ANXA11 sequence in bacterial expression vectors pMCSG7 and pMCSG7-GST. We wanted to know, if the fusion of recombinant protein with GST-tag could reduce fragmentation in the process of protein expression and isolation. After successful test expression of ANXA11 in E. coli strain BL21[DE3] pLysS, we continued with ANXA11 production on a higher scale. Proteins were isolated using Ni2+- or glutathione-S-transferase affinity chromatography. While ANXA11 without GST tended to show some fragmentation, probably due to its flexible N-terminal tail, ANXA11 with GST seemed to be more stable. We successfully cleaved His-GST tag from ANXA11-GST protein with protease TEV. We finally purified recombinant ANXA11 with size-exclusion chromatography.
|
