(143f) Methodology for Redesigning the Specificity of Secreted Proteases
AIChE Annual Meeting
2013
2013 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Protein Engineering II: Techniques II
Monday, November 4, 2013 - 2:00pm to 2:18pm
Proteases constitute 2% of the human genome and regulate many biological processes including cell growth and migration, inflammation, blood coagulation, and programmed cell death. It is estimated that most proteases have on the order of 10-100 physiological substrates, though few studies have elucidated the molecular mechanisms by which proteases achieve a high level of substrate selectivity. A generally applicable, high-throughput strategy to engineer proteases to cleave a target substrate with high specificity and high catalytic efficiency would greatly expand the use of proteases for analytical, biotechnological, and therapeutic applications. We have developed a cell-based assay for redesigning protease selectivity by screening protease mutant libraries for cleavage of a fluorogenic peptide substrate exhibiting Förster resonance energy transfer (FRET). Our screening method relies on using fluorescence activated cell sorting (FACS) for detecting cleavage of the FRET reporter substrate, which will allow screening of large protease mutant libraries (~107members) to identify the activity of interest. As a model system, these novel methods were applied to the protease human kallikrein 7 (hK7) to identify variants that selectively cleave the central hydrophobic core of the amyloid beta (Aβ) peptide, involved in Alzheimer’s disease pathology.
Using Saccharomyces cerevisiae, an expression system was designed and constructed to produce active hK7 and a FRET substrate probe intracellularly. Expression of correctly folded, active hK7 was detected in the yeast cell lysate using an hK7 FRET reporter substrate and confirmed by Western blot. FRET probe production in yeast was measured using flow cytometry and an average 8-fold increase in yellow fluorescence over background was observed after expression for 16 hours at 30°C. We optimized expression conditions to detect hK7 activity in yeast with a co-expressed Aβ8 (KLVF↓F↓AED) FRET substrate using flow cytometry. No activity was detected for yeast cells co-expressing an inactive hK7 mutant or the FRET substrate alone. hK7 displays modest activity towards the target Aβ8 substrate but prefers tyrosine (Y) at the P1 position. We hypothesize that amino acid substitutions around and in the active site may yield variants that prefer the phenylalanine (F) at P1 of Aβ8 and exclude tyrosine, thereby narrowing the specificity towards Aβ8. We randomly mutated the hK7 gene using error-prone PCR to generate two libraries. These libraries were then screened for enhanced activity and selectivity towards the therapeutically relevant target substrate Aβ8 using FACS. This methodology can be applied to engineer other human proteases for highly specific degradation of proteins implicated in a disease state.