Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

Yuki Horiuchi,Jun-Ichi Hotta,Hideaki Mizuno,Danai Laskaratou,Cyril Ruckebusch,Michel Sliwa,Kuniyuki Hatori

doi:10.3390/ijms19020371

Abstract

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Highlights

Fluorescent proteins have become a valuable tool as a reporter system for visualizing detailed structure of cells since the first demonstration of green fluorescent protein (GFP) expression and observation with fluorescence microscopy in Caenorhabditis elegans [1,2]
The frame-insensitive expression cloning vector pRSET-TriEX was made by inserting the DNA sequence for the transcriptional slip14 together with restriction enzyme sites (SmaI, ClaI, and SalI) into the BamHI site of the original vector pRSET B (Figure 2)
To evaluate the effect of the transcriptional slip, the gene encoding eGFP with A, B, or C frame was inserted into pRSET-TriEX and subjected to

Summary

Introduction

Fluorescent proteins have become a valuable tool as a reporter system for visualizing detailed structure of cells since the first demonstration of green fluorescent protein (GFP) expression and observation with fluorescence microscopy in Caenorhabditis elegans [1,2]. The technique relies on the ON/OFF blinking properties [8] found in many fluorescent proteins screened from wild creatures, or mutated from existing ones [9,10]. Efficient rational design of mutants using the knowledge of protein structure has become feasible [11,12]. There are several ways to clone a fluorescent p2roof t1e0in gene from cDmNutAatepdoofrlosmsyenxtishtientgizeodnesfro[9m,10m]. Csleoqnuinegnfcreomwwithildkcnroeawtunreflsuisorescent proteinsst[i1ll4e–ss1e6n]t.iaTlhtoisacmqueitrheondo,vtehl ofluogrhes,ciesnnt optroatpeipnsli[c1a3b].leThteoreexarterascetvienrgalawlaoyws tosecqlounenacfelusoirmesicleanrtity gene from a pcDroNteiAn gpenoeoflr.omExcpDrNesAsipoonolsclsoynntihnegtiziesdafnroamltmerRnNaAti.vTehewmaoystteofffiicniedntawgayenisepeolnycmoedrainsegchflauinorescent protein(rse)acwtioitnh(PuCnRk)n, uoswinng psreiqmueersndceessigfnreodmbaasecdDoNn Athepsoimoill.arTithyeofeanmtiirneo pacoidolseoqfuceDncNe wAitfhroknmowmnRNA is fluorescent proteins [14,15,16]. This method, though, is not applicable to extracting a low sequence introdusciemdiltaoriteyxgperneesfsrioomn avecDctNoArspaonodl.

Methods

Results

Conclusion