RRC ID 3801
Author Kondo N, Ebihara A, Ru H, Kuramitsu S, Iwamoto A, Rao Z, Matsuda Z.
Title Thermus thermophilus-derived protein tags that aid in preparation of insoluble viral proteins.
Journal Anal Biochem
Abstract 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.
Volume 385(2)
Pages 278-85
Published 2009-2-15
DOI 10.1016/j.ab.2008.10.050
PII S0003-2697(08)00771-9
PMID 19084492
MeSH Genomics / methods Green Fluorescent Proteins HIV-1 / chemistry Human Immunodeficiency Virus Proteins / analysis Humans Molecular Probe Techniques Thermus thermophilus / chemistry* Viral Proteins / analysis*
IF 2.877
Times Cited 3
DNA material Thermus thermophilus expression plasmids TEx12F01 (THR004921) TEx18D04 (THR007276)