| y |
| n/a |
| n/a |
| n/a |
| 526 |
| 56180.4 |
| 5.04594 |
| 56180.4 |
| 3 |
MGALARALLLPLLAQWLLRAAPELAPAPFTLPLRVAAATNRVVAPTPGPGTPAERHADGL
ALALEPALASPAGAANFLAMVDNLQGDSGRGYYLEMLIGTPPQKLQILVDTGSSNFAVAG
TPHSYIDTYFDTERSSTYRSKGFDVTVKYTQGSWTGFVGEDLVTIPKGFNTSFLVNIATI
FESENFFLPGIKWNGILGLAYATLAKPSSSLETFFDSLVTQANIPNVFSMQMCGAGLPVA
GSGTNGGSLVLGGIEPSLYKGDIWYTPIKEEWYYQIEILKLEIGGQSLNLDCREYNADKA
IVDSGTTLLRLPQKVFDAVVEAVARASLIPEFSDGFWTGSQLACWTNSETPWSYFPKISI
YLRDENSSRSFRITILPQLYIQPMMGAGLNYECYRFGISPSTNALVIGATVMEGFYVIFD
RAQKRVGFAASPCAEIAGAAVSEISGPFSTEDVASNCVPAQSLSEPILWIVSYALMSVCG
AILLVLIVLLLLPFRCQRRPRDPEVVNDESSLVRHRWK
|
| Like all A1 aspartic proteases, BACE2 is synthesized as a zymogen. The precursor forms are catalytically inactive towards their natural substrates, due to their N-terminal pro-sequence folding across the active site. Activation of aspartic proteases requires the removal of the pro-sequence either in an autocatalytic manner or by the assistance of an activation protease. The activation pathway is characterized by structural changes in a stepwise fashion and has been mapped, in part, by determining the crystal structures of various activation intermediates. Further to the function of regulating the activity of a protease, pro-sequences are known to impact on protease folding, particularly by lowering the free energy of the transition state. The important role of the pro-sequence for folding of aspartic proteases is reflected by the fact that recombinant expression of renin and cathepsin D in mammalian cells decreases significantly in the absence of their pro-sequences. Moreover, all members of this protein family require their pro-sequence for proper folding, with the exception of BACE1, which, although less efficient, does fold in the absence of its pro-sequence. |
| Ostermann N, Eder J, Eidhoff U, Zink F, Hassiepen U, Worpenberg S, Maibaum J, Simic O, Hommel U, Gerhartz B.
(2006)
J Mol Biol,
13,
249-61 |
| Crystallography,Structural Studies |
| N-terminal T7 tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 37.0 |
| n/a |
| pro-BACE2 |
| E. coli cells BL21(DE3)pLysS harboring the respective pro-BACE2 expression plasmid were induced with 0.5 mM IPTG. Cells were resuspended in 50 mM Tris–HCl (pH 8.0) and ruptured by sonication. After centrifugation of the homogenate at 16,500g for 15 min, the inclusion body-containing pellet was washed twice with the same buffer and then dissolved in 50 mM Tris–HCl (pH 8.0), 8 M guanidinium chloride, 30 mM dithioerythritol (DTE). |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| None |
| Washing inclusion body |
| insoluble |
| Dilution |
| 50 mM Tris–HCl (pH 8.0) |
| 50 mM Tris–HCl (pH 8.0), 8 M guanidinium chloride, 30 mM dithioerythritol (DTE) |
| 3 M guanidinium chloride, 0.7 M arginine (pH 10.4), 0.5 mM oxidized glutathione, 1 mM reduced glutathione |
| Washing inclusion body |
| no |
| 10.4 |
| 4.0 |
| n/a |
| 2-3 days |
| GSH/GSSG |
| 1.0/0.5 mM,1.0/0.5 mM,1.0/0.5 mM,1.0,0.5 mM,1.0/0.5 mM,1.0/0.5 mM,1.0/0.5 mM,1.0/0.5 mM,1.0/0.5 mM |
| Pro-BACE2 was refolded by diluting the clear supernatant at a ratio of 1:20 (v/v) into 3 M guanidinium chloride, 0.7 M arginine (pH 10.4), 0.5 mM oxidized glutathione, 1 mM reduced glutathione. After incubation for 16 h at 4 deg. C, the solution was further diluted (20-fold) by addition of 0.7 M arginine (pH 9.4), 1 M NaCl, 0.5 mM oxidized glutathione, 1 mM reduced glutathione. Refolding of pro-BACE2 was completed after incubation for two to three days at 4 deg. C. To generate mature BACE2, we introduced an autoactivation step prior to purification, to avoid the aggregation of pro-BACE2. Autoactivation was achieved by adjusting the pH of the protein solution to 3.2 with citric acid (final concentration 5–10 mM) and hydrochloric acid. The precipitated protein was removed by centrifugation and the pH value of the clear supernatant adjusted to 6.8 by addition of Tris (final concentration 20 mM) and NaOH. Purification was started by adding ammonium sulfate to a final concentration of 1.5 M to apply the refolded protein to a butyl Sepharose column. BACE2 was eluted in a single step with 20 mM Tris–HCl (pH 6.8), containing 350 mM NaCl and subsequently concentrated for size-exclusion chromatography (SEC). To complete autoactivation, the pH of the protein solution was again adjusted to 3.2 and the precipitated protein was removed by centrifugation. The activated BACE2 was then purified by gel-filtration chromatography using a 26/60 Superdex 75 column equilibrated with 10 mM Tris–HCl (pH 6.8), 350 mM NaCl. |
| Enzyme activity,Gel filtration chromatography |
| None |
| L-Arginine |
| 0.7 M |
| 100 mg/l |
| n/a |
| A two-step procedure was developed to solubilize and refold the enzyme as described in Materials and Methods, which led to minimal precipitation of protein during the refolding procedure. However, analysis of the refolded material by size-exclusion chromatography revealed that most of the enzyme aggregated in the form of soluble, high molecular mass protein complexes. This protein form was not able to autoactivate, possibly due to the formation of wrong intra- and intermolecular disulfide bonds. To further optimize the refolding conditions, we have used high concentrations of salt (up to 2 M NaCl) and have performed the refolding at various pH values (7.0 - 9.5). Under these conditions, we have observed a significant portion of the refolded protein to be monomeric. Monomeric pro-BACE2 can be activated by shifting the pH to 3.4. |