fulltext.study @t Gmail

An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography

Paper ID Volume ID Publish Year Pages File Format Full-Text
34684 45039 2012 7 PDF Available
Title
An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography
Abstract

Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20 kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10 kDa- and 20 kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5 kDa-SC-mPEG. Bioactivities of 10 kDa- and 20 kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.

► In this study a solid phase PEGylation method by IMAC was developed. ► Purification and PEGylation process were integrated in one IMAC operation. ► Conjugates via solid phase were more homogeneous than that via liquid phase ones. ► IMAC-assisted PEGylation could protect N-terminal of His-trSak from PEGylation.

Keywords
PEGylation; Staphylokinase; Solid phase; Immobilized metal-ion affinity chromatography
First Page Preview
An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography
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
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 47, Issue 1, January 2012, Pages 106–112
Authors
, , , , , , , ,
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