 | | | | pMAL Protein Fusion and Purification System |
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Prices are in US dollars and valid only for US orders.
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- Reliable expression: substantial yields (up to 100 mg/L) in more than 75% of the cases tested
- Expression in either the cytoplasm or periplasm: periplasmic expression enhances folding of proteins with disulfide bonds
- Fusion to MBP has been shown to enhance the solubility of proteins expressed in E. coli (7)
- Gentle elution with maltose: no detergents or harsh denaturants
Description:
In the pMAL™ Protein Fusion and Purification System, the cloned gene is inserted into a pMAL vector down-stream from the malE gene, which encodes maltose-binding protein (MBP). This results in the expression of an MBP-fusion protein (1,2,3). The technique uses the strong Ptac promoter and the translation initiation signals of MBP to express large amounts of the fusion protein. The fusion protein is then purified by a one-step affinity purification specific for MBP (Figure 1) (4).
The system uses the pMAL vectors which are designed so that insertion interrupts a lacZα gene allowing a blue-to-white screen for inserts on X-gal (5). The vectors include a sequence coding for the recognition site of a specific protease. This allows the protein of interest to be cleaved from MBP after purification, without adding any vector-derived residues to the protein (6). For this purpose, the polylinker includes a restriction site superimposed on the sequence coding for the site of the specific protease. This is where the gene of interest is inserted.
Expression from the pMAL vectors yields up to 100 mg fusion protein from a liter of culture (Figure 2). In most cases, the expressed protein is soluble, as fusion to MBP has been proven to enhance the solubility of proteins expressed in E. coli (7). While no expression system works with every cloned gene, the pMAL™ Protein Fusion and Purification System gives substantial yields of protein in more than 75% of the cases tested. A chapter in Current Protocols in Molecular Biology (3) provides an in-depth analysis of the use of the pMAL vectors.
Figure 1: Schematic of the pMAL System.
Figure 2: SDS-polyacrylamide gel electrophoresis of fractions from the purification of MBP-paramyosin-ΔSal. A: Lane 1: uninduced cells. Lane 2: induced cells. B: Lane 1: purified protein eluted from amylose column with maltose. Lane 2: purified protein after Factor Xa cleavage. Lane 3: paramyosin fragment eluted from second amylose column.
Kit Components:
E. coli K12 TB1
Amylose Resin
Anti-MBP Antiserum
Factor Xa Protease
MBP2* Protein
MBP2*-paramyosinΔSal
pMAL-c4X Vector
pMAL-p4X Vector
Storage Conditions
Storage Temperature:
-20°C
Notes
General notes:- Expected Results: Expression from the pMAL vectors yields up to 100 mg fusion protein from a liter of culture. While no expression system works with every cloned gene, the pMAL™ Protein Fusion and Purification System gives substantial yields of protein in about 75% of the cases tested. A chapter in Current Protocols in Molecular Biology (3) provides an in-depth analysis of the use of the pMAL vectors. The System's instruction manual is available separately upon request.
FAQs
- Where can I find many more detailed FAQs for Protein Fusion & Purification (pMAL) System?
- What are the differences among the various pMAL vectors?
- What are some of the possible explanations for an inability to clone an insert into a pMAL vector?
- My fusion protein is insoluble; is there anything I can do to get it expressed as soluble protein when using the pMAL system?
- What primers should I use to sequence the ends of my insert after I clone it into a pMAL vector?
- When using the pMAL purification system, I see my intact fusion protein by SDS-PAGE when I run cells boiled in sample buffer, but when I check the crude extract the fusion is degraded.
- Much of my fusion protein flows through the amylose column. Is there anything I can do to improve my fusion's affinity for the amylose column when using the pMAL system?
- How many times can I use the amylose column for pMAL purifications?
- How should I store my protein after it is purified with the pMAL system?
- What is MBP2* supplied in the pMAL Purification Kit? Is it different from wild-type MBP produced from E. coli?
References
- Guan, C., Li, P., Riggs, P.D. and Inouye, H. (1987) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene, 67, 21-30.
- Maina, C.V., Riggs, P.D., Grandea, A.G. III, Slatko, B.E., Moran, L.S., Tagliamonte, J.A., McReynolds, L.A. and Guan, C. (1988) Gene, 74, 365-373.
- Riggs, P.D. (1990) In Expression and Purification of Maltose-Binding Protein Fusions. F.M. Ausebel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology, pp. 16.6.1-16.6.10.
- Kellerman, O.K. and Ferenci, T. (1982) W.A. Wood (Eds.), Methods Enzymol., 90, pp. 459-463. New York: Academic Press.
- Yanisch-Perron, C., Vieira, J. and Messing J. (1985) Gene, 33, 103-119.
- LaVallie, E.R. and McCoy, J.M. (1990) F.M. Ausebel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology, pp. 16.4.1-16.4.17.
- Kapust and Waugh (1999) Protein Sci., 8, 1668-1674.
Reagents Sold Separately
E. coli K12 TB1
Amylose Resin
Anti-MBP Antiserum
Factor Xa Protease
MBP2* Protein
MBP2*-paramyosinΔSal
pMAL-c4X Vector
pMAL-p4X Vector
Companion Products
malE Primer (24-mer)
Amylose Magnetic Beads
Amylose Resin High Flow
Anti-MBP Magnetic Beads
Anti-MBP Monoclonal Antibody
Anti-MBP Monoclonal Antibody HRP Conjugated
Enterokinase, light chain
Genenase I
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH PURPOSES ONLY. For commercial use of this system or any components thereof, both Non-Profit and For-Profit buyers and users may obtain a license from New England Biolabs, Inc. U.S. Patent No. 5,643,758.
* pMAL-c2E or pMAL-p2E vectors are covered by U.S. Patent No. 4,769,326 and foreign counterparts owned by the Reagents of the University of California. This product is for research use only and is not to be used for commercial purposes. Use of this product to produce products for sale or for diagnostic, therapeutic or drug discovery purposes is prohibited. In order to obtain a license to use this product for commercial purposes, contact the Regents of the University of California.
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