TY - JOUR
T1 - Expression of a soluble truncated Vargula luciferase in Escherichia coli
AU - Hunt, Eric A.
AU - Moutsiopoulou, Angeliki
AU - Broyles, David
AU - Head, Trajen
AU - Dikici, Emre
AU - Daunert, Sylvia
AU - Deo, Sapna K.
PY - 2017/4/1
Y1 - 2017/4/1
N2 - Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
AB - Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
KW - Bacterial expression
KW - Bioluminescence
KW - Truncated protein
KW - Vargula luciferase
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U2 - 10.1016/j.pep.2017.01.007
DO - 10.1016/j.pep.2017.01.007
M3 - Article
C2 - 28108349
AN - SCOPUS:85011298965
VL - 132
SP - 68
EP - 74
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -