Chaperone-aided in vitro renaturation of an engineered E1 envelope protein for detection of anti-Rubella virus IgG antibodies.
The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.
Scholz
Christian
C
Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany. christian.scholz@roche.com
Thirault
Laurence
L
Schaarschmidt
Peter
P
Zarnt
Toralf
T
Faatz
Elke
E
Engel
Alfred Michael
AM
Upmeier
Barbara
B
Bollhagen
Ralf
R
Eckert
Barbara
B
Schmid
Franz Xaver
FX
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D015870:Gene Expression; D007074:Immunoglobulin G / Q000276:immunology*; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D012412:Rubella virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D012995:Solubility; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000914:Antibodies, Viral; D004220:Disulfides; D007074:Immunoglobulin G; D018832:Molecular Chaperones; D010446:Peptide Fragments; D014759:Viral Envelope Proteins; C040901:rubella antibodies; C061826:E1 envelope protein, Rubella virus