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Efficient soluble expression of two copies of EMP1 connected in series in Escherichia coli, with enhanced EPO activity

Paper ID Volume ID Publish Year Pages File Format Full-Text
34324 45018 2015 7 PDF Available
Title
Efficient soluble expression of two copies of EMP1 connected in series in Escherichia coli, with enhanced EPO activity
Abstract

•Soluble EMP1 dimer was expressed in recombinant E. coli as two-EMP1s-in-series.•Fusion partner thioredoxin could facilitate to fold more effectively than DsbA.•The erythropoietic activity of EMP1 dimer was 10-fold higher than that of monomeric EMP1.•The length of linker peptide had negligible effect on the erythropoietic activity.

Erythropoietin mimetic peptide 1 (EMP1) is a 20-amino-acid cyclic peptide that can increase its erythropoietic activity approximately 10–100 times through dimerization. In the present work, an EMP1 dimer was expressed in a recombinant Escherichi coli system by fusing two EMP1 genes linked by thioredoxin (TrxA) or disulfide bond formation protein A (DsbA). Both of them were also linked with a His6 tag to simplify the subsequent purification processes. The resulting fusion protein, two-EMP1s-in-series, was a homodimer connected by a polyglycine linker of variable length. The results showed that thioredoxin facilitated the expression and folding of two-EMP1s-in-series more properly than DsbA. The highest soluble expression levels were 0.83 g/L for TrxA-EMP1-EMP1 (S), 0.82 g/L for TrxA-EMP1-EMP1 (L) and 0.43 g/L for DsbA-EMP1-EMP1 (S). After being released from the fusion protein construct by enterokinase cleavage and subsequent reversed-phase chromatographic purification, the erythropoietic activity of two-EMP1s-in-series was 10-fold higher than that of monomeric EMP1. The length of the linker peptide between the two EMP1 peptides had a negligible effect on the erythropoietic activity.

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Keywords
EMP1; Erythropoietin mimetic peptide; Thioredoxin; Fusion expression; Soluble expression
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Efficient soluble expression of two copies of EMP1 connected in series in Escherichia coli, with enhanced EPO activity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 5, May 2015, Pages 689–695
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