Expression and characterization of N-terminal domain of Epstein-Barr virus latent membrane protein 2A in Escherichia coli.
Latency of Epstein-Barr virus (EBV) is maintained by the transmembrane protein latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. LMP2A contains a cytoplasmic N-terminal domain composed of 119 amino acids, which provides signals that are responsible for the association with various signal molecules, resulting in negative regulation of B-cell signaling and the EBV lytic cycle. In the present study, to obtain N-terminal domain of LMP2A (LMP2A NTD, 13 kDa) in Escherichia coli for structural analysis, a strategy for obtaining the unfused form of LMP2A NTD without any fusion partners was proposed. Recombinant LMP2A NTD has previously been expressed using the GST fusion system in E. coli [Virology 268 (2000) 178, J. Virol. 71 (1997) 4752, Mol. Cell. Biol. 20 (2000) 8526]. However, we were unable to obtain untagged LMP2A NTD from this construct because of rapid proteolysis by thrombin. To overcome the proteolysis by thrombin, C-terminal His-tagged LMP2A NTD and intein-fused LMP2A NTD were prepared. As a result, LMP2A NTD without a fusion partner could be successfully obtained using non-enzymatic cleavage. The secondary structure of the recombinant LMP2A NTD was analyzed using circular dichroism. In aqueous solution, LMP2A NTD adopts an unordered structure, which was not affected by varying pH and salt concentration. In addition, any secondary structural components of LMP2A NTD were not induced in the membrane-mimicking environments, suggesting that LMP2A NTD may intrinsically have a random coil-like structure. The biological activity of recombinant LMP2A NTD was monitored by chemical shift perturbation in HSQC spectra of LMP2A NTD with or without WW domains, which result supports that the structural change induced by WW domains is restricted within narrow region.
Park
Sung Jean
SJ
National Research Laboratory (MPS), Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Republic of Korea.
Seo
Min-Duk
MD
Lee
Suk Kyeong
SK
Ikeda
Masato
M
Longnecker
Richard
R
Lee
Bong Jin
BJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000821:virology*; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins