One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography

Abstract

We have developed a one-step facile, flexible and readily scalable purification method for a recombinant protein, TM 1-99 (113 amino acid residues; 12,837 Da) based on reversed-phase highperformance liquid chromatography (RP-HPLC) from an E. coli cell lysate. Following cell lysis, the cell contents were extracted with 0.1% aqueous trifluoroacetic acid (TFA), applied directly under conditions of high sample load to a narrow bore RP-HPLC C 8 column (150 mm × 2.1 mm I.D.) and eluted by a shallow gradient of acetonitrile (0.1%/min). Loads of 23 and 48 mg of lyophilized crude cell extract produced 2.4 and 4.2 mg of purified product (>94% pure), respectively, at >94% recovery. Our results show the excellent potential of one-step RP-HPLC for purification of recombinant proteins from cell lysates, where high yields of purified product and greater purity are achieved compared to affinity chromatography. Such an approach was also successful in purifying just trace levels (<0.1% of total contents of crude sample) of TM 1-99 from a cell lysate.

Extract from the Zooxanthellate Jellyfish Cotylorhiza tuberculata Modulates Gap Junction Intercellular Communication in Human Cell Cultures

“…ACN precipitation allows efficiently precipitation of abundant proteins (usually larger than ~40 kDa) in complex biological samples [94,95,96]. Due to its physicochemical properties, ACN is also widely used as solvent in the extraction of high value products; furthermore, salting-out as well as sugaring-out procedures were also used to trigger phase separation in ACN-water mixtures [97,98]. The natural presence of salts in the jellyfish samples allowed a spontaneous phase separation, yielding an upper layer that was primarily ACN (Upper Phase, UP in Figure 3), as described in Mathies and Austin [99], and a partitioning of the proteins in two fractions: Intermediate and Lower Phases (IP and LP), respectively (Figure 3).…”

Identification of a Unique “Stability Control Region” that Controls Protein Stability of Tropomyosin: A Two-stranded α-Helical Coiled-coil

“…This was initially useful for purification of the expressed TM constructs by chromatography using a T7 peptide affinity column. Subsequently and during the progress of this study however, a one-step reversed-phase HPLC procedure was developed and adapted in this laboratory for this purpose 98 .…”

Critical interactions in the stability control region of tropomyosin

“…Eluent A was aqueous 0.2% TFA and eluent B was 0.2% TFA in acetonitrile (Chen et al, 2006; Mills et al, 2006). The gradient initially proceeded from 0-30%B over 15 minutes (2%B per minute) but was followed by a shallower gradient from 30%-55%B over 166 minutes (0.15%B per minute).…”