Thermostabilization of firefly luciferase by a single amino acid substitution at position 217

Abstract

Random mutagenesis of the luciferase cDNA from "Genji" firefly, Luciola cruciata, was induced by hydroxylamine in an attempt to isolate thermostable mutants. Three mutants were isolated, and the cDNAs encoding these proteins were sequenced. All mutant cDNAs carried the same C to T transition mutation that conferred an amino acid substitution of Thr by Ile at position 217. The wild-type luciferase and the thermostable variant (Thr217Ile) were purified to homogeneity, and their enzymatic properties were determined. Thr217Ile was superior to wild-type in thermal and pH stability. Furthermore, the specific activity of the Thr217Ile mutant was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of luciferase, we replaced the Thr residue at position 217 with all of the rest of the possible amino acid residues by site-directed mutagenesis. The thermostability of these substitution mutants seemed to correlate with the hydrophobicity of the substituted amino acid residue.

Mutagenesis of firefly luciferase shows that cysteine residues are not required for bioluminescence activity

“…The fact that PpC216V/K141I retains activity suggests that Cys 216 is not essential for activity, unless a loss of activity due to the C216V substitution has been compensated by the K141I substitution. Interestingly, replacements of the adjacent Thr 217 with isoleucine in Lc luciferase [22] and Ala 217 with either isoleucine, leucine or valine in LI luciferase [23] have been found to increase thermal stability of the enzymes. Mutagenesis has also been shown to cause mutant luciferases to produce different colors of light [24,25] and PpL luciferase to lose activity when the 12 C-terminal amino acid residues are removed [26,27].…”

Thermostabilization of Firefly Luciferases Using Genetic Engineering

“…Especially, Thr217Leu and Thr217Val luciferases still retained over 75% of activity after 10 min incubation at 50°C. The fact that hydrophilic and large hydrophobic (Trp and Tyr) substitution resulted in an expression yield >1000-fold less than wild-type is also consistent with residue 217 being in a buried, hydrophobic environment (Kajiyama & Nakano, 1993). The amino acid sequences of L. cruciata and L. lateralis luciferases are 94% identical.…”

“…10), four of these substitutions were suggested to be the key mutations that cause the high stability of the mutant 4TS: R211L, A217V, E356K, and S364C. The mutations of the residues A217 (Kajiyama & Nakano, 1993) and E356 are known to significantly increase the thermostability of firefly luciferases according to the www.intechopen.com previous studies. The effect of the residues R211 and S364 on thermostability is identified for the first time.…”

“…Firefly luciferase is particularly suitable for activity screens owing to the ease with which its bioluminescence activity can be detected . Kajiyama and Nakano were the first to use random mutagenesis in an attempt to isolate thermostable mutants of the luciferase from L. cruciata fireflies (Kajiyama & Nakano, 1993). The mutagen-treated plasmid was transformed into E.coli JM101.…”

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

“…10,11 Nevertheless, the poor thermostability of wild-type (WT) PhRED, which is also a common problem for all other beetle luciferases 23 and can affect the detection efficiency of in vivo BLI, 29 limits its application in demanding circumstances. Some attempts have been made to improve firefly luciferase thermostability by mutagenesis (e.g., Ppy, [30][31][32] Lcr, 33 Luciola lateralis (Lla), 34 Hotaria parvula (Hpa) 35 ). Among these, mutations of T217 (Lcr) and A217 (Lla) (i.e., I212 of PhRED) to the three most hydrophobic residues (I, V, and L) produced mutants with greater thermostability.…”