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:
-
Strong T7 promoter for high-level, or Rhamnose promoter for tunable, recombinant protein expression.
-
Increased solubility and fast protein purification from N-terminal 6xHis SUMO fusion tag
-
Small size (2.5 kb) for easier downstream manipulation.
-
Patented CloneSmart® technology increases cloning efficiency.
|  |
| 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. |
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.
|  |
| 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
gene number | Soluble protein yield |
| 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°C. 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.
Note: The SUMO protein tag included in these kits is a specially engineered version of SUMO that can be cleaved only using Lucigen's SUMO Express Protease. SUMO Express Protease does not cleave the wild type SUMO substrate at any useful level. Therefore, it is not recommended to use SUMO Expresso Protease to cleave wild type SUMO.
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.
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:
-
Strong T7 promoter for high-level, or Rhamnose promoter for tunable, recombinant protein expression.
-
Increased solubility and fast protein purification from N-terminal 6xHis SUMO fusion tag
-
Small size (2.5 kb) for easier downstream manipulation.
-
Patented CloneSmart® technology increases cloning efficiency.
| |
| 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. |
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.
| |
| 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
gene number | Soluble protein yield |
| 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°C. 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.
Note: The SUMO protein tag included in these kits is a specially engineered version of SUMO that can be cleaved only using Lucigen's SUMO Express Protease. SUMO Express Protease does not cleave the wild type SUMO substrate at any useful level. Therefore, it is not recommended to use SUMO Expresso Protease to cleave wild type SUMO.
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.
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