Abstract
To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t 0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t 0.5 of ~6.0 days in 50 mM HEPES, but ~42 days in 10 mM sodium borate with >85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t 0.5 of PAAS was ~14 days in 50 mM Tris–HCl, and ~21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 °C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.
Abbreviations
- CIAP:
-
Calf intestinal alkaline phosphatase
- ELISA:
-
Enzyme-linked-immunoabsorbent-assay
- HEPES:
-
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
- PAAS:
-
Pseudomonas aeruginosa arylsulfatase
- PAGE:
-
Non-denaturing polyacrylamide gel electrophoresis
- SDS-PAGE:
-
Sodium dodecyl sulfate-PAGE
- t 0.5 :
-
The period for the activity of an enzyme to be 50 % of its initial one during storage
- Tris:
-
Tri-hydroxymethylaminomethane
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Acknowledgments
Supported by National Natural Science Foundation of China (Nos. 30200266, 31570862, 81172965 and 81321004), the Sciences and Technology Commission of Yuzhong District of Chongqing (No. 20130135), the Education Ministry of China (No. 20125503110007), PUMC Youth Foundation (3332015166), and Chongqing Sciences and Technology Commission (CSTC2011BA5039).
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Yuwei Li, Xiaolan Yang have contributed equally to this work.
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Li, Y., Yang, X., Wang, D. et al. Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase. Protein J 35, 283–290 (2016). https://doi.org/10.1007/s10930-016-9671-0
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DOI: https://doi.org/10.1007/s10930-016-9671-0