Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications.
Gaussia luciferase (Gluc)-with its many favorable traits such as small size, bright emission, and exceptional stability-has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed.
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.|University of Miami, Department of Chemistry, Coral Gables, 33146, USA.
Eric A
EA
Hunt
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.|University of Miami, Department of Chemistry, Coral Gables, 33146, USA.
Angeliki
A
Moutsiopoulou
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.
Stephanie
S
Ioannou
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.
Katelyn
K
Ahern
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.
Kristen
K
Woodward
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.
Emre
E
Dikici
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.
Sylvia
S
Daunert
University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry &Molecular Biology, Miami, 33136, USA.
Sapna K
SK
Deo
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002942:Circular Dichroism; D033342:Copepoda / Q000201:enzymology*; D004926:Escherichia coli; D017930:Genes, Reporter; D006207:Half-Life; D008156:Luciferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D017384:Sequence Deletion; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry
D011994:Recombinant Proteins; D014443:Tyrosine; D008156:Luciferases