title: Rapid analysis of protein expression and solubility with the SpyTag-SpyCatcher system.
abstract: Successful isolation of well-folded and active protein often first requires the creation of many constructs. These are needed to assess the effects of truncations, insertions, mutations, and the presence and position of different affinity tags. Determining which constructs yield the highest expression and solubility requires the investigator to express and partially purify each construct, and, in the case of low-expressing proteins, to follow the protein using time-consuming Western blots. Even then, many proteins form soluble aggregates, which may only be apparent after more extensive purification via size exclusion chromatography. In this work, we have utilized a covalent bond-forming tag/domain pair, known as SpyTag/SpyCatcher, to rapidly and specifically attach a fluorescent label to proteins of interest in cellular lysates. Once labeled, tagged proteins can easily be followed via SDS-PAGE and fluorescence size exclusion chromatography (F-SEC) to assess expression levels, solubility, and monodispersity without the need for purification. These techniques enable rapid and facile analysis of proteins, which may greatly facilitate optimization of protein expression constructs.
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States.
authors.forename: Dustin
authors.initials: D
authors.lastname: Dovala
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States.
authors.forename: William S
authors.initials: WS
authors.lastname: Sawyer
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rath
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States. Electronic address: louis.metzger_iv@novartis.com.
authors.forename: Louis E
authors.initials: LE
authors.lastname: Metzger
mesh_terms: D018829:Adhesins, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D033903:Periplasmic Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C470631:FbaB protein, Streptococcus pyogenes; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; C104888:Spy protein, E coli
keywords: Dispersity; Expression test; Solubility; SpyCatcher; SpyTag; Western