A microscale platform for integrated cell-free expression and activity screening of cellulases.
Recent advances in production of cellulases by genetic engineering and isolation from natural microbial communities have necessitated the development of high-throughput analytical technologies for cellulase expression and screening. We have developed a novel cost-effective microscale approach based on in vitro protein synthesis, which seamlessly integrates cellulase expression with activity screening without the need for any protein purification procedures. Our platform achieves the entire process of transcription, translation, and activity screening within 2-3 hours in microwell arrays compared with days needed for conventional cell-based cellulase expression, purification, and activity screening. Highly sensitive fluorescence-based detection permits activity screening in volumes as low as 2-3 μL with minimal evaporation (even at temperatures as high as 95 °C) leading to two orders of magnitude reduction in reagent usage and cost. The platform was used for rapid expression and screening of β-glucosidases (BGs) and cellobiohydrolases (CBHs) isolated from thermophilic microorganisms. Furthermore, it was also used to determine optimum temperatures for BG and CBH activities and to study product inhibition of CBHs. The approach described here is well suited for first-pass screening of large libraries to identify cellulases with desired properties that can subsequently be produced on a large scale for detailed structural and functional characterization.
Chandrasekaran
Aarthi
A
Joint BioEnergy Institute, Emeryville, CA 94608, USA.
Bharadwaj
Rajiv
R
Park
Joshua I
JI
Sapra
Rajat
R
Adams
Paul D
PD
Singh
Anup K
AK
D001426:Bacterial Proteins; D002474:Cell-Free System / Q000378:metabolism*; D044602:Cellulases / Q000032:analysis* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D057166:High-Throughput Screening Assays; D046228:Microarray Analysis; D014176:Protein Biosynthesis; D001617:beta-Glucosidase
D001426:Bacterial Proteins; D044602:Cellulases; D001617:beta-Glucosidase; D043366:Cellulose 1,4-beta-Cellobiosidase