Abstract
BackgroundA superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila.ResultsIn vivo fluorescence spectroscopic analyses of clones carrying a codon-adapted and 6 × His tagged TtsfGFP cassette showed approximately 2–4-fold increased fluorescence emission compared with the control groups at 3 h. Fluorescence microscopy also revealed that TtsfGFP reached its emission maxima at 100 min, which was much earlier than controls expressing EGFP and sfGFP (240 min). A T. thermophila ATP-dependent DNA ligase domain containing hypothetical gene (H) was cloned into the 3' end of 6 × His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 × His-TtsfGFP-H clone confirmed its localization in the macro- and micronucleus of vegetative T. thermophila. Simultaneous affinity purification of TtsfGFP and TtsfGFP-H with Ni-NTA beads was feasible, as shown by Ni-NTA purified proteins analysis by SDS-PAGE and western blotting.ConclusionsWe successfully codon adapted the N-terminal 6 × His-TtsfGFP tag and showed that it could be used for protein localization and affinity purification simultaneously in T. thermophila. We believe that this dual tag will advance T. thermophila studies by providing strong visual traceability of the target protein in vivo and in vitro during recombinant production of heterologous and homologous proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0137-9) contains supplementary material, which is available to authorized users.
Highlights
A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP, is more soluble, faster folding, and is the brightest of the known GFP mutants
To reduce the cost and experimental time of T. thermophila studies, this study aimed to develop an advanced dual fluorescence tag based on a T. thermophila codon-adapted superfolder GFP (sfGFP) and an affinity tag such as 6 × His for simultaneous protein localization and affinity purification of the desired fusion protein
We demonstrated that the T. thermophila codon preferences used in the 6 × His-to create a codon-adapted sfGFP tag (TtsfGFP) tag clearly improved its translational efficiency, resulting in an increase in the emitted fluorescence compared with the control groups (Figure 5)
Summary
A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila. HI069813.1 and Synthetic Sequence 3 from Patent EP1853717] was developed, which has superior features among GFP mutants, such as higher solubility, brighter fluorescence, faster folding, and higher resistance to denaturants such as urea and formamide [12]. These biochemical properties are attributed to the introduction of enhanced GFP mutations (F64L and S65T), cycle-3 GFP mutations (F99S, M153T, and V163A), and Super folder GFP mutations (S30R, Y39N, N105T, Y145F, I171V, and A206V) [12,13,14]. The introduction of a short affinity sequence, such as polyhistidine, to GFP resolves the lack of an affinity tag [16,17,18,19,20]
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