Back to Search
Start Over
Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2014; Vol. 1118, pp. 97-108. - Publication Year :
- 2014
-
Abstract
- We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.
- Subjects :
- Cell-Free System
Enzyme Stability
Escherichia coli cytology
Fungal Proteins chemistry
Fungal Proteins genetics
Fungal Proteins metabolism
Gene Library
Hydrolysis
Lipase chemistry
Lipase genetics
Lipase metabolism
Mutation
Polymerase Chain Reaction
Substrate Specificity
Temperature
Transaminases chemistry
Transaminases genetics
Transaminases metabolism
Triglycerides metabolism
Enzyme Assays methods
Escherichia coli metabolism
Fungal Proteins biosynthesis
Lipase biosynthesis
Protein Biosynthesis
Transaminases biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 1118
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 24395411
- Full Text :
- https://doi.org/10.1007/978-1-62703-782-2_6