Thermus thermophilus-derived protein tags that aid in preparation of insoluble viral proteins.
The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 (T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.
Kondo
Naoyuki
N
Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo, Japan.
Ebihara
Akio
A
Ru
Heng
H
Kuramitsu
Seiki
S
Iwamoto
Aikichi
A
Rao
Zihe
Z
Matsuda
Zene
Z
D023281:Genomics / Q000379:methods; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000737:chemistry; D054298:Human Immunodeficiency Virus Proteins / Q000032:analysis; D006801:Humans; D015336:Molecular Probe Techniques; D016963:Thermus thermophilus / Q000737:chemistry*; D014764:Viral Proteins / Q000032:analysis*
D054298:Human Immunodeficiency Virus Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins