Chaperone-Aided in Vitro Renaturation of an Engineered E1 Envelope Protein for Detection of Anti-Rubella Virus IgG Antibodies

Abstract

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.

Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

“…The peptidyl-prolyl isomerase was beneficial for the refolding of Fab fragments [ 278 ], whilst the disulfide isomerase, alone or in combination with quiescin-sulphydryl oxidase and glutaredoxin, facilitated the oxidative folding of ribonuclease A and riboflavin binding protein [ 279 - 281 ]. The fusion to the chaperone SlyD had a positive effect on the refolding of the ectodomain E1 of Rubella virus [ 282 ]. DsbA, peptidyl-prolyl-isomerase, and GroEL minichaperone were immobilized on an agarose gel to refold the scorpion toxin Cn5 [ 283 ], immobilized DsbA, DsbC and GroEL minichaperone were effective in refolding single-chain fragments [ 284 ], disulfide isomerase was beneficial for ribonuclease and lysozyme refolding [ 285 ], and DnaK in combination with trigger factor or disulfide isomerase helped the functional folding of scFv alone and fused to a toxin domain [ 286 , 287 ].…”

NMR Solution Structure of SlyD from Escherichia coli: Spatial Separation of Prolyl Isomerase and Chaperone Function

“…It might explain why in biotechnological applications aggregation-prone proteins are kept soluble in a very efficient manner when they are linked with two or even three copies of SlyD. 8,9 How does SlyD ⁎ kinetically prevent aggregation? At least for one standard chaperone substrate, namely insulin, we could show that SlyD ⁎ binds early at a time corresponding to the lag phase of nonchaperoned aggregation.…”

“…Even highly aggregation-prone proteins can be functionally solubilized when fused to one or two SlyD modules. 8,9 The PPIase activity of SlyD resides in the FKBP domain and amounts to catalytic efficiencies (k cat / K M ) of 250,000 M − 1 s − 1 (Supplementary Table 1) when assessed with short peptide substrates such as Suc-Ala-Xaa-Pro-Phe-pNA. 7 Remarkably, the activity increases about 4-fold with large protein substrates such as the disulfide-reduced and Scarboxymethylated form (RCM) of ribonuclease T1 (RNase T1 with Ser54 and Pro5 replaced by Gly and Asn, respectively)";(RCM-T1; with Ser54 and Pro5 replaced by Gly and Asn, respectively) as long as the IF domain is present.…”

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

“…Indeed, covalent fusion of aggregation-prone proteins to one or two C-terminally truncated SlyD modules results in enhanced cytosolic expression and solubility. 26,27 SlyD, 26,28 FkpA, [29][30][31] trigger factor, [32][33][34] SurA, 35 and MtFKBP17 36 exhibit both prolyl isomerase activity and chaperone activity. In most of these proteins, enzymatic and chaperone-like activities are localized to separate domains.…”