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Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO

Marblestone, Jeffrey G. ; Edavettal, Suzanne C. ; Lim, Yiting ; Lim, Peter ; Zuo, Xun ; Butt, Tauseef R.

Protein science, 2006-01, Vol.15 (1), p.182-189 [Periódico revisado por pares]

Bristol: Cold Spring Harbor Laboratory Press

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Título:
Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO
Autor: Marblestone, Jeffrey G. ; Edavettal, Suzanne C. ; Lim, Yiting ; Lim, Peter ; Zuo, Xun ; Butt, Tauseef R.
Assuntos: Cloning, Molecular - methods ; Cysteine Endopeptidases - biosynthesis ; Cysteine Endopeptidases - chemistry ; Cysteine Endopeptidases - genetics ; DUB, deubiquitinating enzyme or ubiquitin specific protease/hydrolase ; eGFP, enhanced green fluorescent protein ; Endopeptidases - biosynthesis ; Endopeptidases - chemistry ; Endopeptidases - genetics ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; fusion protein ; Gene Fusion ; Humans ; IPTG, isopropropyl‐β‐D‐thiogalactopyranoside ; MBP, E. coli maltose‐binding protein ; Ni‐NTA, nickel‐nitrilotriacetic acid ; PCR, polymerase chain reaction ; protease ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis ; Solubility ; structural genomics ; SUMO ; SUMO-1 Protein - biosynthesis ; SUMO-1 Protein - chemistry ; SUMO-1 Protein - genetics ; Ub, ubiquitin ; ubiquitin‐like protein ; Ubl(s), ubiquitin‐like protein(s) ; ULP, catalytic domain of Ulp1
É parte de: Protein science, 2006-01, Vol.15 (1), p.182-189
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Reprint requests to: Tauseef R. Butt, LifeSensors Inc., 271 Great Valley Parkway, Malvern, PA 19355, USA; e-mail: butt@lifesensors.com; fax: (610) 644-8616.
Descrição: Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult‐to‐express proteins remains empirical. To determine which fusion tags are best suited for difficult‐to‐express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green florescent protein (eGFP), matrix metalloprotease‐13 (MMP13), and myostatin (growth differentiating factor‐8, GDF8), were fused to the C termini of maltose‐binding protein (MBP), glutathione S‐transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructswere expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similarKM values, but SUMOprotease had a 25‐fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.
Editor: Bristol: Cold Spring Harbor Laboratory Press
Idioma: Inglês

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Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO

Marblestone, Jeffrey G. ; Edavettal, Suzanne C. ; Lim, Yiting ; Lim, Peter ; Zuo, Xun ; Butt, Tauseef R.

Bristol: Cold Spring Harbor Laboratory Press

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