Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protein of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.
Keywords: CBD; Cell harvesting; Cell lysis; Chitin beads; IMPACT™-System; Intein/Chitin binding protein tags; Protein affinity purification.
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To access the quality of our sample, both the commercial insulin (Insulin Injection, MW 5778) and the B22D desB30 insulin analog was subjected to size exclusion chromatography (SEC) using Superdex 75 (GE healthcare) column under standard conditions.
Purification of DPIP by the IMPACT-TWIN system. a: Scheme of the protein expression and purification. b: E. coli strain BL21 (DE3) transformed with pTWIN1-DPIP was cultured, induced with IPTG and fractionated to purify the recombinant fusion protein. Fractions were analyzed by 12% SDS–PAGE. M: MW marker (97, 66, 43, 31, 24, 14 kDa), 1: E coli lysate before induction, 2: lysate after induction, 3: soluble fraction after induction. 4: redissolved inclusion body before dialysis, 5: redissolved fraction after dialysis, 6: redissolved, dialyzed fraction flow through. An arrow marks the full-length fusion protein
B22Asp desB30 insulin
Rate of CI-DPIP cleavage increases then slows down after two days. a: SDS-PAGE analysis of cleavage with different incubation time. Lanes 1–3 indicate resin after incubation of 6 days, 4 days and 2 days, respectively, and lane 4 indicates the input precursor without any cleavage (0d). Since the precursor is chemically stable in solution, so Lane 4 shows no CBD + intein fraction, M: molecular weight markers. b: Quantified cleavage ratio. All assays were carried out in triplicate and the average was used in this study; the error bars show standard deviations
The eluate from intein splicing was digested by trypsin at 30 °C for one hour to obtain the final B22D desB30 insulin analog. We used an enzyme/substrate ratio of 1:200 (w/w). The mixture was subsequently purified by high-pressure liquid chromatography (Wasters) (C8 column, acetonitrile and trifluoroacetic acid (TFA) as mobile phases). Monomeric B22D insulin analog was eluted at 19.5-min mark with a flow rate of 1 ml/min and a buffer H gradient of 35–50% over 30 min.
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