- Email: [email protected]
Chloroform phase partitioning for purification of plant-expressed proteins
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 344 (2005) 150–151 www.elsevier.com/locate/yabio
Notes & Tips
Chloroform phase partitioning for puriWcation of plant-expressed proteins K. Boonrod ¤, M. Moser, G. Krczal Centrum Grüne Gentechnik, Rlp-Agroscience GmBH, Breitenweg 71, D-67435 Neustadt, Germany Received 14 March 2005 Available online 8 June 2005
Plant-expressed proteins can usually be directly puriWed from the plant sap by using diVerent techniques according to their physico-chemical properties. Column chromatography is a straightforward method, because it is easy and fast. However, direct puriWcation of plantexpressed proteins from plant sap can lead to the obstruction of the column by insoluble debris such as plant organelles and green pigments. Accumulation of this debris in the column reduces the puriWcation Xow rate. Precipitation of plant-expressed proteins by organic solvents such as acetone, ethanol, methanol, or trichloroacetic acid is time consuming [1–3]. In addition, resuspension of the proteins after precipitation may change the conformation of the target proteins. We describe here a method to separate soluble recombinant proteins from insoluble plant debris and green pigments rapidly, simply, and reproducibly by applying a chloroform (CHCl3) extraction step prior to the proper puriWcation process. CHCl3 and CHCl3/methanol mixtures have been successfully used to extract periplasmic proteins from bacteria [4] and membrane proteins from plants [5], respectively. To analyze whether cytoplasmatic proteins can be isolated from crude plant extract using these methods, 100 mg of Nicotiana benthamiana leaves of a transgenic plant expressing the green Xuorescent protein GFP16C1 [6] were homogenized in 100 l of total soluble protein extraction buVer (1% Triton X-100 in phosphate-buVered saline). After centrifugation (5 min, 14,000 rpm), the supernatant was phase partitioned with either 100 l CHCl3, methanol, or a CHCl3/methanol *
1
Corresponding author. Fax: +49 0 6321 671313. E-mail address: [email protected] (K. Boonrod). Abbreviation used: GFP, green Xuorescent protein.
0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.05.014
mixture (1:1). The samples were centrifuged at 14,500 rpm for 5 min. The clear, water phase of each sample was transferred into a new tube and exposed to UV light to monitor GFP Xuorescence. Our results show that the plant sap extracted with CHCl3 alone shows Xuorescence but not when extracted with methanol or the CHCl3/methanol mixture (data not shown). This indicated that the GFP activity was impaired when methanol was used for extraction. We speculate that methanol aVects the protein structure probably by inducing conformational changes of the molecule. We further tested the applicability of this rapid extraction method for the puriWcation of other proteins. We focused our attention on a single-chain antibody, expressed via a viral vector. In detail, a bivalent singlechain (bi-scFv) fragment against a seed-speciWc transcription factor, FUS3 [7], and scFvD9, a single chain against urease [8] containing myc-tag at C terminal, were expressed by an infectious tomato bushy stunt virusderived clone, FC8 (unpublished data). In vitro transcripts of the viral RNAs carrying anti-FUS3-bi-scFv gene and scFvD9 gene were inoculated on N. benthamiana leaves, respectively; 5 days postinoculation the infected leaves were harvested and extracted with total soluble protein extraction buVer. The proteins were then phase partitioned with CHCl3 as described above. The water phase was taken and an aliquot of this fraction was subjected to a 10% SDS–PAGE for Western blot analysis. To demonstrate that the CHCl3-extracted proteins corresponded to the recombinant proteins, the samples containing bi-scFv and scFvD9 were puriWed by using a protein-L column (Actigen). After washing and elution, 10 l of each fraction was subjected to 10% SDS–PAGE. The Western blot analysis shows that the anti-FUS3-biscFv and scFvD9 were puriWed by phase partitioning
Notes & Tips / Anal. Biochem. 344 (2005) 150–151
151
data revealed that CHCl3 phase partitioning of all soluble plant proteins tested was faster than standard protocols. We present a simple and rapid method for separating the green pigments and cell debris from total soluble protein extracts. Thus, CHCl3 can be used to avoid obstruction of CHCl3 of chromatography column during puriWcation processes. CHCl3 extraction aVected neither enzyme activity nor binding activity of the proteins examined.
Acknowledgments We thank Dr. Udo Conrad and Dr. Grudrun Mönke for anti-FUS3-scFv gene and anti-myc antibody and Dr. Jean-Pierre Mach for scFvD9 gene. We also thank Steven Heaps and Dr. Michael Wassenegger (CGG, Neustad) for reviewing the manuscript.
References
Fig. 1. Western blot and enzyme-linked immunosorbent assay (ELISA) analysis displaying successful puriWcation of the bi-scFv and scFvD9-His. Total soluble protein containing anti-FUS3-biscFv or scFvD9 was extracted with CHCl3. After centrifugation the water phase was loaded into protein-L columns; 10 l of each eluted fraction was subjected to 10% SDS–PAGE. The presence of scFvs was detected as described. (A) Lane 1 is a negative control; lanes 2 and 3 are the elutants of bi-scFv and scFv9, respectively. M is a protein marker. (B) Determination of the binding activity of the puriWed anti-FUS3-bi-scFv; 0.2 g of puriWed non-CHCl3-treated and CHCl 3-treated anti-FUS3-bi-scFv was applied for an ELISA assay as described [8]. Column 1 is the binding activity of puriWed nonCHCl 3-treated scFvs as a positive control; column 2 is the binding activity of puriWed CHCl3-treated scFvs; column 3 is a negative control (bovine serum albumin; BSA).
with CHCl3 (Fig. 1A). Moreover, the binding activity of the puriWed scFvs was retained when it was tested by enzyme-linked immunosorbent assay (Fig. 1B); 15 g puriWed proteins was obtained from 1 g of infected leaves material. This method results in 50% yield increase compared to puriWcation without CHCl3 treatment. Our
[1] L. Jiang, L. He, M. Fountoulakis, Comparison of protein precipitation methods for sample preparation prior to proteomic analysis, J. Chromatogr. A 1023 (2004) 317–320. [2] O.Y. Dmitriev, K. Altendorf, R.H. Fillingame, Subunit a of the E. coli ATP synthase: reconstitution and high resolution NMR with protein puriWed in a mixed polarity solvent, FEBS Lett. 556 (2004) 35–38. [3] D. Wessel, U.I. Flugge, A method for quantitative recovery of proteins in dilute solution in the presence of detergence and lipids, Anal. Biochem. 138 (1984) 141–143. [4] G. Ferro-Luzzi Ames, C. Prody, S. Kustu, Simple, Rapid and quantitative release of periplasmic proteins by chloroform, J. Bacteriol. 160 (1984) 1181–1183. [5] D. Seigneurin-Berny, N. Rolland, J. Garin, J. Joyard, DiVerential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-speciWc proteomic approach to identify rare intrinsic membrane proteins, Plant J. 19 (1999) 217–228. [6] M.T. Ruiz, O. Voinnet, D.C. Baulcombe, Initiation and maintenance of virus-induced gene silencing, Plant Cell 10 (1998) 937–946. [7] G. Monke, L. Altschmied, A. Tewes, W. Reidt, H.P. Mock, H. Baumlein, U. Conrad, Seed-speciWc transcription factors ABI3 and FUS3: molecular interaction with DNA, Planta 219 (2004) 158–166. [8] M. Houimel, I. Corthesy-Theulaz, I. Fisch, C. Wong, B. Corthesy, J. Mach, R. Finnern, Selection of human single chain Fv antibody fragments binding and inhibiting helicobacter pylori urease, Tumor Biol. 22 (2001) 34–36.
Notes & Tips
Chloroform phase partitioning for puriWcation of plant-expressed proteins K. Boonrod ¤, M. Moser, G. Krczal Centrum Grüne Gentechnik, Rlp-Agroscience GmBH, Breitenweg 71, D-67435 Neustadt, Germany Received 14 March 2005 Available online 8 June 2005
Plant-expressed proteins can usually be directly puriWed from the plant sap by using diVerent techniques according to their physico-chemical properties. Column chromatography is a straightforward method, because it is easy and fast. However, direct puriWcation of plantexpressed proteins from plant sap can lead to the obstruction of the column by insoluble debris such as plant organelles and green pigments. Accumulation of this debris in the column reduces the puriWcation Xow rate. Precipitation of plant-expressed proteins by organic solvents such as acetone, ethanol, methanol, or trichloroacetic acid is time consuming [1–3]. In addition, resuspension of the proteins after precipitation may change the conformation of the target proteins. We describe here a method to separate soluble recombinant proteins from insoluble plant debris and green pigments rapidly, simply, and reproducibly by applying a chloroform (CHCl3) extraction step prior to the proper puriWcation process. CHCl3 and CHCl3/methanol mixtures have been successfully used to extract periplasmic proteins from bacteria [4] and membrane proteins from plants [5], respectively. To analyze whether cytoplasmatic proteins can be isolated from crude plant extract using these methods, 100 mg of Nicotiana benthamiana leaves of a transgenic plant expressing the green Xuorescent protein GFP16C1 [6] were homogenized in 100 l of total soluble protein extraction buVer (1% Triton X-100 in phosphate-buVered saline). After centrifugation (5 min, 14,000 rpm), the supernatant was phase partitioned with either 100 l CHCl3, methanol, or a CHCl3/methanol *
1
Corresponding author. Fax: +49 0 6321 671313. E-mail address: [email protected] (K. Boonrod). Abbreviation used: GFP, green Xuorescent protein.
0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.05.014
mixture (1:1). The samples were centrifuged at 14,500 rpm for 5 min. The clear, water phase of each sample was transferred into a new tube and exposed to UV light to monitor GFP Xuorescence. Our results show that the plant sap extracted with CHCl3 alone shows Xuorescence but not when extracted with methanol or the CHCl3/methanol mixture (data not shown). This indicated that the GFP activity was impaired when methanol was used for extraction. We speculate that methanol aVects the protein structure probably by inducing conformational changes of the molecule. We further tested the applicability of this rapid extraction method for the puriWcation of other proteins. We focused our attention on a single-chain antibody, expressed via a viral vector. In detail, a bivalent singlechain (bi-scFv) fragment against a seed-speciWc transcription factor, FUS3 [7], and scFvD9, a single chain against urease [8] containing myc-tag at C terminal, were expressed by an infectious tomato bushy stunt virusderived clone, FC8 (unpublished data). In vitro transcripts of the viral RNAs carrying anti-FUS3-bi-scFv gene and scFvD9 gene were inoculated on N. benthamiana leaves, respectively; 5 days postinoculation the infected leaves were harvested and extracted with total soluble protein extraction buVer. The proteins were then phase partitioned with CHCl3 as described above. The water phase was taken and an aliquot of this fraction was subjected to a 10% SDS–PAGE for Western blot analysis. To demonstrate that the CHCl3-extracted proteins corresponded to the recombinant proteins, the samples containing bi-scFv and scFvD9 were puriWed by using a protein-L column (Actigen). After washing and elution, 10 l of each fraction was subjected to 10% SDS–PAGE. The Western blot analysis shows that the anti-FUS3-biscFv and scFvD9 were puriWed by phase partitioning
Notes & Tips / Anal. Biochem. 344 (2005) 150–151
151
data revealed that CHCl3 phase partitioning of all soluble plant proteins tested was faster than standard protocols. We present a simple and rapid method for separating the green pigments and cell debris from total soluble protein extracts. Thus, CHCl3 can be used to avoid obstruction of CHCl3 of chromatography column during puriWcation processes. CHCl3 extraction aVected neither enzyme activity nor binding activity of the proteins examined.
Acknowledgments We thank Dr. Udo Conrad and Dr. Grudrun Mönke for anti-FUS3-scFv gene and anti-myc antibody and Dr. Jean-Pierre Mach for scFvD9 gene. We also thank Steven Heaps and Dr. Michael Wassenegger (CGG, Neustad) for reviewing the manuscript.
References
Fig. 1. Western blot and enzyme-linked immunosorbent assay (ELISA) analysis displaying successful puriWcation of the bi-scFv and scFvD9-His. Total soluble protein containing anti-FUS3-biscFv or scFvD9 was extracted with CHCl3. After centrifugation the water phase was loaded into protein-L columns; 10 l of each eluted fraction was subjected to 10% SDS–PAGE. The presence of scFvs was detected as described. (A) Lane 1 is a negative control; lanes 2 and 3 are the elutants of bi-scFv and scFv9, respectively. M is a protein marker. (B) Determination of the binding activity of the puriWed anti-FUS3-bi-scFv; 0.2 g of puriWed non-CHCl3-treated and CHCl 3-treated anti-FUS3-bi-scFv was applied for an ELISA assay as described [8]. Column 1 is the binding activity of puriWed nonCHCl 3-treated scFvs as a positive control; column 2 is the binding activity of puriWed CHCl3-treated scFvs; column 3 is a negative control (bovine serum albumin; BSA).
with CHCl3 (Fig. 1A). Moreover, the binding activity of the puriWed scFvs was retained when it was tested by enzyme-linked immunosorbent assay (Fig. 1B); 15 g puriWed proteins was obtained from 1 g of infected leaves material. This method results in 50% yield increase compared to puriWcation without CHCl3 treatment. Our
[1] L. Jiang, L. He, M. Fountoulakis, Comparison of protein precipitation methods for sample preparation prior to proteomic analysis, J. Chromatogr. A 1023 (2004) 317–320. [2] O.Y. Dmitriev, K. Altendorf, R.H. Fillingame, Subunit a of the E. coli ATP synthase: reconstitution and high resolution NMR with protein puriWed in a mixed polarity solvent, FEBS Lett. 556 (2004) 35–38. [3] D. Wessel, U.I. Flugge, A method for quantitative recovery of proteins in dilute solution in the presence of detergence and lipids, Anal. Biochem. 138 (1984) 141–143. [4] G. Ferro-Luzzi Ames, C. Prody, S. Kustu, Simple, Rapid and quantitative release of periplasmic proteins by chloroform, J. Bacteriol. 160 (1984) 1181–1183. [5] D. Seigneurin-Berny, N. Rolland, J. Garin, J. Joyard, DiVerential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-speciWc proteomic approach to identify rare intrinsic membrane proteins, Plant J. 19 (1999) 217–228. [6] M.T. Ruiz, O. Voinnet, D.C. Baulcombe, Initiation and maintenance of virus-induced gene silencing, Plant Cell 10 (1998) 937–946. [7] G. Monke, L. Altschmied, A. Tewes, W. Reidt, H.P. Mock, H. Baumlein, U. Conrad, Seed-speciWc transcription factors ABI3 and FUS3: molecular interaction with DNA, Planta 219 (2004) 158–166. [8] M. Houimel, I. Corthesy-Theulaz, I. Fisch, C. Wong, B. Corthesy, J. Mach, R. Finnern, Selection of human single chain Fv antibody fragments binding and inhibiting helicobacter pylori urease, Tumor Biol. 22 (2001) 34–36.