A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process.
Rubredoxin (Rub) from Thermotoga maritima, a 6.1-kDa red protein containing an Fe(III)-cysteine(4) center, was evaluated for its usefulness as a colored fusion tag for expression of recombinant proteins in E. coli. Here, we describe the Rub features relevant to accelerating screening for optimal high yield soluble expression conditions and automating the ensuing purification process. Spectroscopic properties and the yield of Rub fused to a typical target protein were compared to analogous GFP and Flavodoxin constructs, showing Rub absorption to be sufficient for structural genomics purposes while being produced at much higher soluble levels than GFP constructs. Based entirely on Rub absorption at 380 nm, both generic and affinity purification of crude cell lysate were performed: thus guided anion exchange purification of a Rub fusion construct as well as automated Ni-NTA purification resulted in pure protein. Rub is stable over a wide range of pH, temperature, and buffer environments, enabling robust purification protocols. Across a variety of fusion constructs, including N- and C-terminal Rub, quantitation via the Rub signal was shown to reliably correlate with analytical HPLC data obtained at 220 nm. We propose the "RubyTag" as an alternative to conventional protein fusion tags, as it combines a specific absorption signal with convenient biochemical and biological properties. Further, it allows direct on-line readout on conventional chromatography systems, holding promise for automated multi-step chromatography.
Kohli
Bernhard M
BM
Biozentrum, Basel, Switzerland.
Ostermeier
Christian
C
D018845:CD11 Antigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005418:Flavodoxin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012416:Rubredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D018845:CD11 Antigens; D005418:Flavodoxin; D008164:Luminescent Proteins; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; D012416:Rubredoxins; D049452:Green Fluorescent Proteins; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid