title: In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling.
abstract: BACKGROUND
  Given the expanding antiretroviral therapy, inexpensive and fast HIV drug resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HIV-1 protease inhibitors (PIs) based on recombinant expression of patient-derived HIV PR in Escherichia coli and subsequent enzymatic testing in a fluorescent readout.
  
  
  OBJECTIVE
  To facilitate and expedite the test procedure, we have introduced coupled in vitro transcription/translation using a commercially available technology called RTS for producing enzymatically active HIV-1 protease (PR).
  
  
  METHODS
  We expressed one wild type PR and one highly resistant mutant starting from molecular clones as well as three patient-derived PRs. The amplified PR gene was either ligated into an expression vector or directly used as a template for the in vitro transcription/translation reaction. Enzymatic susceptibility data derived from in vitro expressed PRs were correlated to the respective results from E. coli expression and genotypic evaluation.
  
  
  RESULTS
  All tested enzymes were obtained in sufficient quantities for complete resistance profiling to five PIs. The PRs required no purification prior to the enzymatic assay. Inhibition constants and enzymatic resistance factors compared well to corresponding data from PRs expressed in parallel in E. coli. Enzymatic resistance was in good agreement with the respective PR genotype.
  
  
  CONCLUSIONS
  The presented in vitro transcription/translation system represents a novel approach for HIV PR expression starting from molecular clones or patient samples. Coupled with the enzyme-kinetic PR assay recently developed in our group it allows to sensitively quantify resistance to PIs. The test system is significantly less laborious and faster than currently available phenotypic drug resistance assays.
authors.affiliation: Department of Virology, Max von Pettenkofer-Institute, University of Munich, Pettenkoferstr. 9a, D-80336 Munich, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Hoffmann
authors.forename: Bernd
authors.initials: B
authors.lastname: Buchberger
authors.forename: Cordula
authors.initials: C
authors.lastname: Nemetz
mesh_terms: D003001:Cloning, Molecular; D024882:Drug Resistance, Viral / Q000235:genetics*; D016333:HIV Protease / Q000096:biosynthesis* / Q000235:genetics; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010641:Phenotype; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
chemical_list: D017320:HIV Protease Inhibitors; D016333:HIV Protease