A Simple Purification Method for Heat-Stable Recombinant Low Molecular Weight Proteins and Peptides Via GST-Fusion Products

Abstract

Here, we describe a simple, rapid, cost-effective, and efficient novel one-step purification method for GST-tagged peptides and small proteins. This novel technique applies to proteins and peptides that are known to be thermally stable at 60 °C and do not have elaborate structure(s) and whose heat-induced unfolding is reversible. This method takes advantage of glutathione S-transferase from Schistosoma japonicum (sj26GST) precipitating when heated at 60 °C. Purified GST-fusion products are subjected to enzymatic cleavage to separate the GST tag from the target peptide or small proteins. In our proposed method, the cleavage products are heated at 60 °C for 20 min which results in the precipitation of the GST tag. Subsequently, the GST tag is separated from the target peptide or small protein by high-speed centrifugation. Biophysical experiments such as SDS-PAGE, circular dichroism, isothermal titration calorimetry, mass spectroscopy, and multidimensional NMR spectroscopy confirm that the target peptides and small proteins are purified to more than 95% homogeneity, intact native conformation, and no significant change in the binding affinity of heat-treated purified product to the interacting partners.

Keywords: Aggregation; GST purification; Heat treatment; Recombinant proteins.

Fig. 1

SDS-PAGE of purification and thrombin cleavage of GST-CD2 (32 kDa): lane-1, pre-stained protein marker; lane-2, pellet post cell lysis; lane-3, supernatant post lysis; lane-4, flow through; lane-5, eluted GST-CD2; lane-6, 8 M urea; lane-7, cleaved GST-CD2 using thrombin

Fig. 2

(a) SDS-PAGEs of heat treatment: lane-1, pre-stained protein marker; lane-2, GST-CD2; lane-3, cleaved GST CD2; lane-4, supernatant 45 °C; lane-5, pellet 50 °C; lane-6, supernatant 50 °C; lane-7, pellet 55 °C; lane-8, supernatant 55 °C; lane-9, pellet 60 °C; lane-10, supernatant 60 °C; lane-11, pellet 65 °C; lane-12, supernatant 65 °C; lane-13, pellet 70 °C; lane-14, supernatant 70 °C; lane-15, pellet 75 °C; lane-16, supernatant 75 °C; lane-17, pellet 80 °C; lane-18, supernatant 80 °C; lane-19, pellet 85 °C; lane-20, supernatant 85 °C. (b) Absorbance of pure GST-tag monitored its aggregation index at temperatures ranging from 40 to 80 °C

Fig. 3

(a) Flow chart comparing conventional purification method and heat treatment method. (b) SDS-PAGE of purification of CD2 (6 kDa) using heat treatment method (lanes-3–4) or conventional purification method (lanes-5–6): lane-1, pre-stained protein marker; lane-2, cleaved GST-CD2 using thrombin; lane-3, pellet after heat treatment; lane-4, supernatant after heat treatment; lane-5, eluted CD2 in flow-through; lane-6, eluted GST with 10 mM reduced GSH. (c) Western blot of heat treatment method: lane-1, GST-CD2; lane-2, cleaved GST-CD2; lane-3, supernatant post-heat treatment, lane-4, pellet post-heat treatment; lane-5, pre-stained protein marker. (d) Yields of CD2: heat treatment method vs. conventional purification method

Fig. 4

(a) SDS-PAGE of purification of PBD46 (5.1 kDa) using the heat treatment method. Lane-1, MWM; lane-2, PBD46 bound to GST; lane-3, PBD46 after cleavage by thrombin; lane-4, 30 °C treatment pellet; lane-5, 30 °C treatment supernatant; lane-6, 50 °C treatment pellet; lane-7, 50 °C treatment supernatant; lane-8, 70 °C treatment pellet; lane-9, 70 °C treatment supernatant; lane-10, 90 °C treatment pellet; lane-11, 90 °C treatment supernatant. (b) DSC spectrum of PBD46 purified by heat treatment. The Tm of the peptide is 41.6 °C (black) which was consistent with the observed Tm of 41.3 °C for PBD46 produced by the conventional double chromatography method (gray). (c) Mass spectrum confirming the size of PBD46. The mass is around 10 kDa because PBD46 is known to dimerize. (d) MALDI-TOF spectrum of PBD46 was analyzed to confirm the amino acid sequence of the peptide

Fig. 5

(a) Tricine gel of purification of AlbM4 peptide (1.5 kDa): lane-1, pellet after lysis; lane-2, supernatant after lysis; lane-3, flow through; lane-4&5, eluted GST-AlbM4; lane-6, cleaved fusion protein; lane-7, pellet after heat treatment; lane-8, supernatant after heat treatment; lane-9, pre-stained ultra-low protein marker. (b) SDS-PAGE of purification of A) S100A13 (11.5 kDa): lane-1, pellet after lysis; lane-2, supernatant after lysis; lane-3, eluted GST-S100A13; lane-4, cleaved GST S100A13; lane-5, pellet after heat treatment; lane-6, supernatant after heat treatment; lane-7, pre-stained protein marker

Fig. 6

(a) SDS-PAGE of purification of WAP (7 kDa): lane-1, pellet after lysis; lane-2, supernatant after lysis; lane-3, eluted GST-WAP; lane-4, cleaved GST WAP; lane-5, pre-stained protein marker; lane-6, WAP. (b) Mass spectrum confirms the size of WAP. (c) SDS-PAGE of purification of the HB-peptide (3.7 kDa): lane-1, pellet after lysis; lane-2, supernatant after lysis; lane-3, flow through; lane-4, eluted GST-HB; lane-5, cleaved GST HB; lane-6, pellet after heat treatment; lane-7, HB-peptide; lane-8, pre-stained protein marker. (d) Mass spectrum confirms the size of HB-peptide

Fig. 7

Isothermal titration calorimetry of (a) heat-treated CD2 vs. 54-peptide (K_d = 1.27 μM) and (b) conventionally purified CD2 vs. 54-peptide (K_d = 1.42 μM)

Fig. 8

(a) Differential scanning thermograms of heat-treated CD2 and GST-CD2. (b) Thermal denaturation of conventionally purified CD2

Fig. 9

(a) Far UV circular dichroism spectra, (b) intrinsic fluorescence emissions spectra and (c) overlay of 2D 1H15N-HSQC spectra and (d) chemical shift perturbation plot of heat-treated and conventionally purified CD2

Fig. 10

Far UV CD spectra of heat-treated AlbM4 and synthetic AlbM4 confirm the characteristic random coil secondary structure