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IMPACT-CN System > FAQ |  | IMPACT-CN System FAQ
See the Gene Expression and Protein Purification FAQ also.
Q1: Where can I find many more detailed FAQs for IMPACT systems?
Q2: What factors contribute to the low expression of some proteins when using IMPACT?
Q3: What are some suggestions for expressing a toxic gene using the IMPACT system?
Q4: How can I use IMPACT with an insoluble fusion protein?
Q5: What are the advantages of the IMPACT System?
Q6: What is the binding capacity of the resin in the IMPACT system?
Q7: If the fusion protein forms inclusion bodies, can I try to purify protein under denaturing conditions using IMPACT?
Q8: Can the chitin beads in the IMPACT kit be regenerated?
Q9: What is the success rate of procaryotic protein purification using the IMPACT system?
Q10: What size range of proteins have been purified with the IMPACT system?
Q1: Where can I find many more detailed FAQs for IMPACT systems?
A1: You may find the complete set of FAQs for IMPACT under "Technical Reference > Expression Systems" on our Web site.
Q2: What factors contribute to the low expression of some proteins when using IMPACT?
A2: Successful expression and purification of a target protein in a pTYB vector depend on the following factors: (a) E. coli strain; (b) culture conditions (e.g., temperature, aeration, cell density); (c) induction and protein expression conditions (temperature, time, IPTG concentration); (d) expression level of the fusion protein; (e) solubility of the fusion protein; (f) efficiency of thiol-induced cleavage; (g) solubility of the target protein after the cleavage.
The expression of the fusion protein in the C-terminal fusion vectors(pTYB1,2,3 and 4) may be primarily determined by the expression level of the N-terminal target protein. In the case of the N-terminal fusion vectors(pTYB11 and 12) the expression level is less dependent on the target protein. It is conceivable that different target proteins, due to certain structural constraints, may prefer either C-terminal or N-terminal fusion to allow proper folding of the fusion precursor and a high level of protein expression. Some target proteins tested (the majority of them eucaryotic proteins) did not express well in E.coli. The use of a eucaryotic expression system may ultimately help to increase the yield and obtain properly modified eucaryotic proteins. The presence of RNA structure that sequesters the translation initiation sequence may decrease the efficiency of translation. Poor codon usage, mRNA degradation or proteolysis may all contribute to poor expression. Different growth and induction conditions should be tested to optimize the expression of the fusion protein. Induction at lower temperatures may reduce the formation of inclusion bodies as well as proteolysis. Protease deficient hosts should be tested to minimize proteolysis. Improved codon usage or translational coupling may be effective in increasing the expression level. Another possibility for poor expression could be that the clone is not stable due to the toxicity of the target protein to the host cells. One should inoculate the medium with a freshly grown colony and induce the expression at lower temperatures with lower IPTG concentrations (0-1 mM). Some target proteins become insoluble after on-column cleavage and therefore are only eluted after incubation of the resin with0.3 N NaOH. In this case, increasing the salt concentration (0.5-2 M NaCl) or adding a nonionic detergent to the Cleavage Buffer may improve the solubility of the target protein.
Q3: What are some suggestions for expressing a toxic gene using the IMPACT system?
A3: Establish the initial construct in a non expression strain (no T7 RNA polymerase gene)
* Incubate the plates and inoculate the culture (from a fresh colony) at 20-30°C (not at 37°C)
* When you are ready to express, try using a strain which is designed for the expression of extremely toxic genes. Use of BL21(DE3)/pLys may result in tighter control of expression. Stratagene has a specific system called Lambda CE6 induction kit which is based on BL21 cells and bacteriophage CE6. In this case T7 RNA polymerase is introduced via CE6. BL21 cells alone do not carry the T7 RNA polymerase gene. For more information see Stratagene catalog 97/98 page 112-113.
* In the case of solubility problems:
1. Induce expression at low temperatures to reduce solubility problems (15°C overnight)
2. Following sonication, check both crude cell extracts and clarified cell extracts by SDS-PAGE and Western analysis to figure out if there is a solubility problem
* Use the intein-mediated protein ligation [See Part 6: Applications/Ligation (IPL) and TWIN] technique to express and purify a truncated, inactive protein and ligate to the missing expressed sequence to restore activity [Evans et. al.,(1998) Protein Sci.7,2256-2264].
Q4: How can I use IMPACT with an insoluble fusion protein?
A4: If you have problems with solubility one of the first things to try is varying the temperature of induction (15 °C or lower; some customers have used 8-12 °C induction temperatures) and/or the concentration of IPTG.
There are some steps which effect solubilization of the protein:
*A fresh colony from an overnight plate is necessary for optimal expression
*Induction conditions - Overnight at 15°C with 0.3 mM IPTG or 30°C for 3 hours is recommended.
*Try lowering the IPTG concentration to 0.01-0.1 mM.
*Resuspend the cell pellet in at least 50 ml column buffer/L culture.
*It is crucial to handle the sonication cautiously. If the protein solution gets warm or the solution foams there is a possibility that proteins are being denatured. Use an ice-chilled water bath to keep the cell suspension cool.
If you wish to use a detergent in your buffer, we recommend 0.1-0.5% Triton X-100 or 0.1-0.2% Tween-20. Too much detergent will impair binding to the chitin column.
When purifying a protein that may be insoluble, several factors should be considered:
1. The binding efficiency of the intein-tag to the chitin resin is lower at 4 M urea or higher.
2. The intein-mediated cleavage reaction may be carried out at 0-2 M urea.
3. The higher the urea concentration, the better the chance to solublize a target protein. However, the cleavage reaction should beperformed in 0-2 M urea.
Suggestions for methods to try to keep your protein soluble during cleavage and elution as well as methods to resolubilize after cleavage can be found in the expanded FAQs under "Technical References → Protein Expression".
Q5: What are the advantages of the IMPACT System?
A5: Single column purification - no additional steps to remove the affinity tag
* Yields native amino acid sequence
* Fusion of a target protein to either the C-terminus or N-terminus of the intein
* Proteases are NOT required to remove the affinity tag from the target protein
* Use of either thiol reagent or pH and temperature shift to induce on-column cleavage
* Isolation of proteins with or without an N-terminal methionine residue
* Production of proteins possessing an N-terminal cysteine and/or C-terminal thioester for use in protein labeling, ligation and cyclization
* T7 Promoter for high-level expression and tight regulation of expression temperatures which might destabilize the target protein
* Protein semisynthesis - The ability to create a thioester at the C-terminus for ligation with a N-terminal containing cysteine peptide or tag
* The ability to label the C-terminus of the target protein
Q6: What is the binding capacity of the resin in the IMPACT system?
A6: Based on the binding assays with the maltose-binding protein (MBP) fusion, 2 mg of MBP protein was released from a 1 ml bed of chitin beads, after cleavage of the MBP fusion protein. The kit includes 20 ml of chitin beads which are supplied as a 38 ml slurry in 20% ethanol. Chitin beads are also sold separately, in both 20 ml (NEB# S6651S) and 100 ml (NEB# S6651L) sizes. Store Chitin Beads at 4°C. Temporary storage at -20°C will not affect the binding capacity.
Q7: If the fusion protein forms inclusion bodies, can I try to purify protein under denaturing conditions using IMPACT?
A7: Purification and cleavage of the C-terminal fusion (MBP-intein-CBD fusion) protein has been tested under various urea concentrations. 1-4 M urea allowed 80% binding efficiency while 5-8 M urea showed 30-50% binding efficiency. The efficiency of cleavage with 30 mM DTT in 2 M urea is at least 50%, in comparison to reactions under native conditions. Based on these data, chitin chromatography and on-column cleavage may be carried out in 1-2 M urea. Since DTT may cause rapid cleavage of the fusion protein under denaturing conditions, following the wash step the column flow should be stopped and the DTT stock solution should be added directly to the column.
Q8: Can the chitin beads in the IMPACT kit be regenerated?
A8: The chitin beads can be regenerated at least 5 times. After stripping the chitin column with 3 volumes of 0.3 M NaOH (Stripping solution). Allow the beads to saok for 30 minutes, wash the resin with additional 7 column volumes of 0.3 M NaOH. Then wash the resin with 20 bed volumes of water followed by 5 column volumes of Column Buffer. For long term storage, add 0.02% sodium azide to the Column Buffer. The beads should be stored at 4°C. Temporary storage at -20°C will not affect the binding capacity.
Q9: What is the success rate of procaryotic protein purification using the IMPACT system?
A9: 80% of the procaryotic proteins tested with the IMPACT System were expressed and purified with yields ranging from 0.5-15 mg/liter culture. 50% of these proteins were purified with their native sequences (no vector derived residues).
Q10: What size range of proteins have been purified with the IMPACT system?
A10: The size range of proteins we have purified from E.coli host cells is 11-124 kDa.
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