Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners.
A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895.
Agro-Environmental Research Institute, National Agricultural Research and Innovation Centre, Department of Applied and Environmental Microbiology, H-1022 Budapest, Hungary.
Sándor
S
Varga
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried (Planegg), Germany.
Ganesh Ramnath
GR
Pathare
Agro-Environmental Research Institute, National Agricultural Research and Innovation Centre, Department of Applied and Environmental Microbiology, H-1022 Budapest, Hungary.
Erzsébet
E
Baka
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried (Planegg), Germany.
Marius
M
Boicu
Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Aquaculture and Environmental Safety, Department of Environmental Safety and Ecotoxicology, H-2100 Gödöllő, Hungary.
Balázs
B
Kriszt
Agro-Environmental Research Institute, National Agricultural Research and Innovation Centre, Department of Applied and Environmental Microbiology, H-1022 Budapest, Hungary.
András
A
Székács
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried (Planegg), Germany.
Luca
L
Zinzula
Agro-Environmental Research Institute, National Agricultural Research and Innovation Centre, Department of Applied and Environmental Microbiology, H-1022 Budapest, Hungary.
József
J
Kukolya
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried (Planegg), Germany. Electronic address: nagy@biochem.mpg.de.
István
I
Nagy
D001105:Archaea / Q000201:enzymology; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D012995:Solubility; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics
D019843:Archaeal Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin
E.coli; Protein expression; Protein purification; Protein solubility; SAMP; Tag removal