title: Escherichia coli-expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen.
abstract: BACKGROUND
  The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies.
  
  
  METHODS
  A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels.
  
  
  RESULTS
  In-frame deletion of three hydrophobic regions, spanning amino acid residues 1-43, 519-538 and 676-706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses.
  
  
  CONCLUSIONS
  This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.
authors.affiliation: Department of Biotechnology, University of Turku, 20520 Turku, Finland. talshe@utu.fi
authors.forename: Sheikh M
authors.initials: SM
authors.lastname: Talha
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Nemani
authors.forename: Teppo
authors.initials: T
authors.lastname: Salminen
authors.forename: Sushil
authors.initials: S
authors.lastname: Kumar
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
authors.forename: Kim
authors.initials: K
authors.lastname: Pettersson
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015483:HIV Antibodies / Q000097:blood*; D019302:HIV Envelope Protein gp160 / Q000235:genetics / Q000302:isolation & purification; D015658:HIV Infections / Q000175:diagnosis*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
chemical_list: D000956:Antigens, Viral; D015483:HIV Antibodies; D019302:HIV Envelope Protein gp160; D011994:Recombinant Proteins; C514797:gp160 protein, Human immunodeficiency virus 1