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Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

Paper ID Volume ID Publish Year Pages File Format Full-Text
2817 136 2016 9 PDF Available
Title
Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life
Abstract

•Trypsin stored at pH 3.0 underwent cold denaturation during freezing.•Trypsin stored at pH 8.2 with cryoprotectants remained native and active.•Trypsin stored at pH 8.2 with cryoprotectants was efficient in TMF.

Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634 cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.

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Keywords
Enzyme activity; Cold stability; Protein recovery; Protein denaturation; Proteolysis; Proteomics
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Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 105, Part A, 15 January 2016, Pages 168–176
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us