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). STUDY DESIGN: 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. CONCLUSION: 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.

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. The possibility of working with linear expression templates instead of cloned plasmids and the ease of downstream processing, circumventing the need for cell-lysis, makes them ideally suited for high-throughput screening applications. Among these novel cell-free systems, the Rapid Translation System (RTS) developed by Roche is the first one that is scalable from micrograms to milligrams of protein. This review describes the basic principles of RTS which differentiate it from traditional in vitro expression technologies, starting from template generation to high-end applications like labeling for structural biology research. Recent results obtained by RTS users from different institutions are presented to illustrate each step of a novel cell-free protein expression workflow and its benefits compared to traditional cell-based expression.

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. Base exchanges immediately adjacent to the start codon up to nucleotide (+)25 had a profound effect on translational efficiency. Correlation analysis revealed a significant dependence on base pair probability and G+C content on the expression level, indicating that mRNA secondary structure in this region hampers translation. Using our training data, we developed a methodology to predict and improve the translation efficiency of open reading frames (ORFs).