Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku,Tokyo 113-8656, Japan
E-mail: nagamune{at}bio.t.u-tokyo.ac.jp
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Abstract |
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Keywords: -helix/fluorescence resonance energy transfer/fusion protein/green fluorescent protein/linker engineering
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Introduction |
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The construction of a fusion protein involves the linking of two proteins or domains of proteins by a peptide linker. The selection of the linker sequence is particularly important for the construction of functional fusion proteins. Several studies have been made on the linker selection (Argos, 1990; Alfthan et al., 1995
; Robinson and Sauer, 1998
; Crasto and Feng, 2000
). These studies suggested mainly that the flexibility and hydrophilicity of the linker were important not to disturb the functions of the domains. However, in our previous study on Streptococcal protein G-Vargula luciferase chimera, simple linking of the two moieties by a flexible linker did not retain the binding activity of protein G C1 domain (Maeda et al., 1997
). The loss of the binding activity of protein G could be envisaged due to some sort of interaction and interference between the two moieties. When a three
-helices bundle domain of Staphylococcal protein A (PA) was introduced between protein G and luciferase as a linker, goat/sheep IgG binding activity derived from protein G was regained. We reasoned that PA could spatially separate protein G from luciferase and thus reduce the interference between them. These results indicate that spatial separation of the hetero-functional domains of a fusion protein by proper linker peptide might be so effective that the domains work independently. However, there have been very few reports on the linkers which effectively separate the domains. Although PA was effective as a linker in our case, the length of more than 50 amino-acid residues and the additional binding activity to IgG might hinder it from general use.
In this study, we designed shorter linkers than PA to effectively separate bifunctional domains of a fusion protein. The linkers, which were expected to form a monomeric hydrophilic -helix, were designed according to the previous study on a short peptide forming a monomeric
-helix (Marqusee and Baldwin, 1987
). In that study, the best helix-forming peptide, AEAAAKEAAAKEAAAKA [(i+4)E,K], which was stabilized by GluLys+ salt bridges, showed ~80% helicity. To our knowledge, there have been no reports that tried to introduce the helix-forming peptide to a bifunctional fusion protein as a linker, and it has been unknown whether it can actually separate the domains. To study these, we designed the linkers of different lengths A(EAAAK)nA (n = 25) based on the helix-forming peptide (i+4)E,K, and introduced them between two domains of a bifunctional fusion protein. We also introduced flexible linkers and PA into the fusion protein for the comparison.
As a model fusion protein, we employed fusion proteins of two Aequorea green fluorescent protein (GFP) variants enhanced blue fluorescent protein (EBFP; F64L, S65T, Y66H, Y145F) (Heim and Tsien, 1996) and enhanced green fluorescent protein (EGFP; F64L, S65T) (Cormack et al., 1996
). EBFP and EGFP are useful for fluorescence resonance energy transfer (FRET) studies (Miyawaki et al., 1997
; Tsien, 1998
; Arai et al., 2000
). Because a FRET signal provides information about distances in the order of 10 to 100 Å, the technique is suitable for investigating spatial relationships of targeted molecules (Wu and Brand, 1994
; Selvin, 1995
). In this study, we examined the relative distance between EBFP and EGFP using FRET in order to evaluate designed linkers which effectively separate domains of a bifunctional fusion protein.
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Materials and methods |
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For the expression of the fusion proteins, pET TRX Fusion System 32 (Novagen, Madison, WI, USA), a fusion expression system with Escherichia coli thioredoxin (Trx) was employed to enhance the solubility of the highly expressed proteins in E.coli cytoplasm (LaVallie et al., 1993). The DNA fragment encoding EBFP was prepared from plasmid pEBFP-N1 (Clontech, Palo Alto, CA, USA) by standard PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) and the primers containing the cleavage sites for EcoRV and HindIII. The amplified fragments were digested and cloned into pET32/VLEGFP (Arai et al., 2000
) between EcoRV and HindIII to give the plasmid pET32/EBFPSLEGFP. DNA sequencing was performed using the fluorescence DNA sequencer SQ-5500 (Hitachi, Tokyo, Japan). The DNA fragments encoding the designed linkers were prepared by annealing and extension using the appropriate oligo-nucleotide primers with the cleavage sites for HindIII and NotI. These were digested and cloned into pET32/EBFPSLEGFP between HindIII and NotI to give the plasmids pET32/EBFPlinkerEGFPs.
The amino acid sequences of the designed linkers were: short linker (SL), LAAA (4 aa) (derived from the cleavage sites for HindIII and NotI); flexible linker 3 (FL3), LGGGGSGGGGSGGGGSAAA (19 aa); flexible linker 4 (FL4), LSGGGGSGGGGSGGGGSGGGGSAAA (25 aa); helical linker 2 (HL2), LAEAAAKEAAAKAAA (15 aa); helical linker 3 (HL3), LAEAAAKEAAAKEAAAKAAA (20 aa); helical linker 4 (HL4), LAEAAAKEAAAKEAAAKEAAAKAAA (25 aa); helical linker 5 (HL5), LAEAAAKEAAAKEAAAKEAAAKEAAAKAAA (30 aa); B domain of Staphylococcal PA, LFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPS-QSANLLAEAKKLNDAQAAA (55 aa).
Protein expression and purification
Escherichia coli AD494(DE3)pLysS (Novagen) was transformed with the plasmids, and selected on LB agar plates containing 50 µg/ml ampicillin, 34 µg/ml chloramphenicol and 15 µg/ml kanamycin. For all the cultivations thereafter, LB medium containing the same concentration of the three antibiotics was used. The medium (1.5 l) was inoculated with 5 ml overnight culture at 30°C of each strain harboring pET32/EBFPlinkerEGFPs containing the designed linkers, and cultured at 30°C for 7 h. At an OD600 of 0.5, IPTG was added to a final concentration of 1 mM to induce the expression of the fusion proteins, and cells were further cultured for 12 h at 16°C. Harvested cells were resuspended in sonication buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7.0) and then disrupted by freezethaws and sonication with a Sonifier 250 (Branson, Danbury, CT, USA). The supernatant was obtained after centrifugation twice at 20000 g for 20 min at 4°C. The fusion proteins which have the (His)6-tag were purified using Talon metal affinity resin (Clontech) according to the manufacturer's protocol. The fractions with sufficient fluorescence activity were collected. The proteins were specifically digested with thorombin (Novagen) between Trx and EBFP, and Trx fragments were removed by size exclusion chromatography with Superdex 75 (Amersham Pharmacia Biotech, Uppsala, Sweden). The protein concentration was determined by BCA assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as a standard.
Measurement of fluorescence intensity and circular dichroism (CD) spectra
The fluorescence intensity of the fusion proteins at 444 nm of the EBFP emission peak was measured using a fluorescence spectrophotometer F-2000 (Hitachi) with 380 nm excitation at 25°C. In order to cut the linkers between EBFP and EGFP domains, the fusion proteins (2 µM) were digested by trypsin (50 nM) at 20°C for 12 h in phosphate-buffered saline (pH 7.4). Specific digestion of the linkers was confirmed by SDSPAGE. The CD spectra of the fusion proteins were measured using a CD spectropolarimeter J-725 (Jasco, Tokyo, Japan) at 25°C.
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Results and discussion |
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The design of linkers is more important if a multi-domain protein is designed de novo. The control of the distance and the orientation of the domains in order to maximize the desired functions is a demanding task in near-future protein engineering. Here we would like to propose a term `linker engineering', whose objective is the rational control of the linker conformation, flexibility and the distance between protein domains to create a multi-functional fusion protein. This study suggests the potential of the helical linkers as first candidates for its building block.
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Notes |
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Acknowledgments |
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References |
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Received February 20, 2001; accepted May 14, 2001.