Soluble expression in Escherichia coli of murine endogenous retroviral transmembrane envelope protein having immunosuppressive activity.
Recently, we cloned a fragment of the endogenous murine leukemia viral envelope gene from beta cell line (MIN6N8a) as a new autoantigen gene, whose product was reactive with nonobese diabetic (NOD) mice sera. As a result of determination of nucleotide sequence, this envelope protein had the CKS-17 peptide sequence having immunosuppressive activity. To investigate the role of our cloned transmembrane envelope protein in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM) as an autoantigen or immunosuppressive modulator, a high amount of transmembrane envelope protein was essentially required. Therefore, the expression of our cloned retroviral transmembrane envelope gene was tried in Escherichia coli by a pET vector. However, the expression rate was very low (less than 2%) and most of the expressed protein was insoluble. To improve solubility, the hydrophobic transmembrane anchor domain of our envelope gene was deleted and then the expression of the hydrophilic transmembrane envelope protein was carried out by using the same pET expression system. The expressed protein was completely soluble and the expression yield was dramatically improved by around 25-fold increase. The hydrophilic transmembrane envelope protein was purified by one-step Ni-NTA affinity chromatography and then the fusion tag consisting of the six-histidine peptide and S peptide was removed by cleavage with enterokinase. The processed hydrophilic retroviral transmembrane envelope protein was still immune reactive with NOD sera and also showed immunosuppressive activity by down-regulating the Th1-type cytokine (interferon-gamma) and up-regulating the Th2-type cytokine (interleukin 10).
Kim
K S
KS
Laboratory of Endocrinology, Institute for Medical Sciences, Suwon, Kyunggi-Do, Korea.
Kim
K H
KH
Choi
S E
SE
Yoon
J W
JW
Kang
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000209:etiology / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007166:Immunosuppressive Agents / Q000302:isolation & purification; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D016688:Mice, Inbred NOD; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D001324:Autoantigens; D017931:DNA Primers; D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D016753:Interleukin-10; D007371:Interferon-gamma