In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling.

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.

Objectives: 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).

Rapid translation system: a novel cell-free way from gene to protein.

Proteome research has recently been stimulated by important technological advances in the field of recombinant protein expression. One major breakthrough was the development of a new generation of cell-free transcription/translation systems. The open and flexible character of these systems allows direct control over expression conditions via the addition of supplements to the expression reaction.

Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system.

The dependence of efficiency of translation initiation on mRNA sequence parameters was investigated in an Escherichia coli in vitro expression system. We designed a large-scale expression experiment focussing on the influence of sequence variations in the translated region (TR) of the mRNA without changing the 5'-untranslated region (5'-UTR). The level of translated protein from 756 expression constructs was measured and the influence of a large number of possible effector attributes was statistically analyzed.