Abstract
To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive l-arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of l-arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 29-41 |
| Number of pages | 13 |
| Journal | Applied Biochemistry and Biotechnology |
| Volume | 151 |
| Issue number | 1 |
| DOIs | |
| State | Published - Oct 1 2008 |
| Externally published | Yes |
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Keywords
- Antiplatelet aggregation
- Gel filtration refolding
- RGD motif
- Refolding
- Reverse dilution refolding
- Staphylokinase
ASJC Scopus subject areas
- Biochemistry
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology
- Molecular Biology
- Environmental Engineering
Cite this
Refolding of a staphylokinase variant Y1-sak by reverse dilution. / He, Jintian; Wang, Gaizhen; Xu, Ruiguang; Feng, Jinlin; Wang, Jinlong; Su, Huabo; Song, Houyan.
In: Applied Biochemistry and Biotechnology, Vol. 151, No. 1, 01.10.2008, p. 29-41.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Refolding of a staphylokinase variant Y1-sak by reverse dilution
AU - He, Jintian
AU - Wang, Gaizhen
AU - Xu, Ruiguang
AU - Feng, Jinlin
AU - Wang, Jinlong
AU - Su, Huabo
AU - Song, Houyan
PY - 2008/10/1
Y1 - 2008/10/1
N2 - To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive l-arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of l-arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.
AB - To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive l-arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of l-arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.
KW - Antiplatelet aggregation
KW - Gel filtration refolding
KW - RGD motif
KW - Refolding
KW - Reverse dilution refolding
KW - Staphylokinase
UR - http://www.scopus.com/inward/record.url?scp=53749086110&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=53749086110&partnerID=8YFLogxK
U2 - 10.1007/s12010-008-8150-2
DO - 10.1007/s12010-008-8150-2
M3 - Article
VL - 151
SP - 29
EP - 41
JO - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
JF - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
SN - 0273-2289
IS - 1
ER -