title: Universal genetic assay for engineering extracellular protein expression.
abstract: A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli . However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli . Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible in situ without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli . Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production.
authors.affiliation: Department of Microbiology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Charles H
authors.initials: CH
authors.lastname: Haitjema
authors.forename: Jason T
authors.initials: JT
authors.lastname: Boock
authors.forename: Aravind
authors.initials: A
authors.lastname: Natarajan
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Dominguez
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Gardner
authors.forename: David H
authors.initials: DH
authors.lastname: Keating
authors.forename: Sydnor T
authors.initials: ST
authors.lastname: Withers
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D000595:Amino Acid Sequence; D044602:Cellulases / Q000235:genetics / Q000378:metabolism; D020633:Cellvibrio / Q000201:enzymology; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; C508772:YebF protein, E coli; D044602:Cellulases