Expresso¢ç SUMO

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Expresso¢ç SUMO Cloning and Expression Systems

Soluble protein has never been this fast and easy!

  • Enzyme-free directional PCR cloning in seconds!
  • Save days of effort with ready-to-use vectors and competent cells - NO ligation step.
  • Tightly-controlled expression of N-terminal 6xHis-tagged proteins with cleavable SUMO solubility tag.
  • Systems available with expression under the control of T7 or tunable Rhamnose promoters.
Please note: commercial entities require a license from Brookhaven Science Associates to purchase T7 product.
 

Complete Cloning and Protein Expression System

The Expresso SUMO Cloning and Protein Expression Systems are designed for fast, easy, and efficient directional cloning and soluble expression of PCR-amplified genes. For proteins that form inclusion bodies or are difficult to express in soluble form, the pETite¢ç N-His SUMO and pRham¢â N-His SUMO Vectors allow expression of target proteins with an amino-terminal 6xHis-SUMO protein tag. SUMO (small ubiquitin-like modifier) is a relatively small (100-residue) polypeptide that has been shown to enhance the soluble expression of many proteins that are otherwise difficult to produce in E. coli.
 
The Expresso SUMO Cloning and Protein Expression Systems are based on the original Expresso T7 and Expresso Rhamnose Cloning and Protein Expression Systems, which use Expressioneering Technology¢â to provide effortless high-efficiency cloning and tightly controlled protein expression. The T7 System is complete with pre-processed pETite N-His SUMO cloning vector, and two competent cell lines, supplied in single transformation vials. High efficiency HI-Control¢â 10G Chemically Competent Cells enable stable cloning and HI-Control BL21(DE3) Competent Cells provide tightly controlled protein expression, thus helping you avoid protein expression problems seen with leaky T7 promoter-based expression plasmid systems. The Rhamnose System is complete with pre-processed pRham N-His SUMO cloning vector and high-efficiency E. cloni 10G Competent Cells, which are used for cloning and expression, enabling higher protein expression throughput. The N-terminal 6xHis SUMO tagged recombinant proteins can be rapidly purified using standard Nickel chromatography. SUMO Express Protease is included to provide efficient cleavage of the SUMO tag, precisely at the junction between the SUMO tag and the target protein.
 
Expressioneering-flow-diagram-long.jpg
 

Five-Second Directional Cloning of PCR-Amplified Genes

The Expresso SUMO Cloning and Protein Expression Systems use Expressioneering Technology, the enzyme-free recombinational cloning strategy of the Expresso system to seamlessly integrate the gene with the expression plasmid. The target gene is amplified by PCR using primers that add 18 base-pairs of vector-complementary sequence to both ends of the gene. Unlike other restriction enzyme based methods or ligase-free cloning methods, no further cleanup or enzymatic treatment of the PCR product is necessary. Simply mix 1 µl of the unpurified PCR reaction with the supplied pre-processed pETite or pRham N-His SUMO expression plasmids, and transform immediately into the Chemically Competent Cells provided (Figure 1). 
 

SUMO Vectors includes:

  1. Strong T7 promoter for high-level, or Rhamnose promoter for tunable, recombinant protein expression.
  2. Increased solubility and fast protein purification from N-terminal 6xHis SUMO fusion tag
  3. Small size (2.5 kb) for easier downstream manipulation.
  4. Patented CloneSmart¢ç technology increases cloning efficiency.

Rescue inclusion bodies and insoluble protein with SUMO protein tag:

We have used the Expresso T7 and Expresso T7 SUMO Cloning and Expression Systems for expression and purification of a variety of proteins. Some results of an ongoing large-scale expression study to identify hydrolase enzymes from Fibrobacter succinogenes are presented in Figure 3. Initially, 48 genes were selected for expression trials and cloned into the pETite T7 C-His Vector. Approximately half of these clones have produced soluble, active hydrolase protein, while in other instances target proteins were expressed in an insoluble form. Five of the genes producing insoluble proteins were reamplified and cloned into the pETite SUMO vector. When the resulting clones were expressed in HI-Control BL21(DE3) cells, recovery of active protein in the soluble fraction was significantly improved in four of the five cases (Table 1). Although tag removal was not necessary for hydrolase activity, the tag could be removed efficiently by SUMO Express Protease.
 
 
 
Vectors-SUMO-web.jpg
Figure 2. SUMO expression vector: Small size (2.5 kb vs. 5.4 kb for pET) for easier manipulation, including targeted mutagenesis.  Expression plasmid is pre-processed for instant enzyme-free cloning of PCR products.
 
 
Expresso-Sumo.jpg
Figure 3. Large-scale cloning and expression case study: (A) PCR products from 48 putative hydrolase genes ranging from ~1 to >3 kb. (B) Uninduced (-) and IPTG-induced (+) samples of HI-Control BL21(DE3) cells with 6 different genes cloned into the pETite C-His Vector. (C) Enhanced solubility of SUMO-tagged 2201 and 2442 gene products. Total cell extract and soluble fractions are shown. (D) Removal of 6xHis-SUMO tag from purified SUMO-2201 fusion protein by SUMO protease. –prot: uncleaved SUMO-2201 fusion protein after IMAC purification; +prot: SUMO protease-treated fusion protein; C: isolated 2201 protein after removal of 6xHis-SUMO fragment and SUMO protease by subtractive IMAC.
 
Fibrobacter succinogenes
Soluble protein yield 
gene number w/o SUMO tag w/SUMO tag
1425 0 mg/liter 0 mg/liter
1765 0 mg/liter 10 mg/liter
1793 0 mg/liter 17 mg/liter
1994 0 mg/liter 17 mg/liter
2201 0 mg/liter 20 mg/liter
 
Table 1.  Improvement of soluble protein yield with SUMO tag. Yield of soluble protein was improved significantly for 4 of 5 Fibrobacter succinogenes genes when cloned into pETite N-His SUMO and expressed in HI-Control BL21(DE3) Cells. Cultures were induced with 1mM IPTG and grown overnight at 22¡ÆC. Yields were calculated from the amount of pure protein obtained from 100 ml of cell culture after purification over a Ni-NTA column.
 

Cleavage of SUMO protein tag

After IMAC purification of the N-His-SUMO tagged protein, the tag portion can be removed precisely by the included SUMO Express Protease. The SUMO Express Protease recognizes the tertiary structure of SUMO rather than a short recognition sequence and cleaves precisely at the junction between the SUMO tag and the target protein, with no off-target cleavage. Both the SUMO Express Protease, which is 6xHis tagged, and the cleaved N-His-SUMO tag can then be separated from the released target protein by subtractive IMAC.
Important Product Use Information
This product is the subject of U.S. Patent #6,709,861. Additional patent applications owned by Lucigen Corporation are pending.
The 6xHis tag is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only for the use in research. Information about licenses for commercial use is available from QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden, Germany. Purification of 6xHis tagged proteins with Ni-NTA manufactured by QIAGEN is highly recommended for best performances and to avoid poor purification results.

SUMO Express Protease is manufactured and supplied by LifeSensors, Inc.

HI-Control BL21(DE3) products are sold under a license issued to Lucigen Corporation by Brookhaven Science Associates, LLC for noncommercial research purposes only. A separate license is required for any commercial use, including the use of these materials for research purposes or production purposes by any commercial entity. Information about commercial licenses may be obtained from the Office of Technology Commercialization and Partnership, Brookhaven National Laboratory, Bldg. 490-C, P. O. Box 5000, Upton, New York 11973-5000, telephone (631)-344-7134. In using this product, you agree that HI-Control BL21(DE3) cells or derivatives containing the cloned gene for T7 RNA Polymerase may not be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of this limited label license and agrees to be bound by its terms.
 
 
 
 
Please note that the SUMO Protease and SUMO Fusion tag are modified from the original native proteins. Native or non-modified control proteins or proteases will not be compatible. For any questions about the SUMO tag or SUMO Protease and associated uses, please contact Lucigen Technical Service. Lucigen SUMO tags are compatible with SUMO Star protease from LifeSensors, but not SUMO Protease I.
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