Expression of SNAP-tag Fusions (N9174)
This plasmid can serve as an expression vector for SNAP-tag fusions or the SNAP-tag alone in appro-priate E. coli strains. The strain needs to provide tightly regulated T7-polymerase in order to achieve expression from the T7 promoter upstream of the gene. It works well in the arabinose inducible strain BL21-AI™ available from Invitrogen Corporation. The pSNAP-tag(T7) is a high copy number expression plasmid that does not contain the lac repressor. Therefore it will not work well in IPTG inducible strains with poorly regulated expression such as BL21 (DE3).
The following instructions have been developed from our experience with the expression of various SNAP-tag fusion proteins and the SNAP-tag. However, if your particular fusion protein requires specific conditions that are not compatible with the ones mentioned below, it is useful to try test expressions and determine whether the SNAP-tag activity is retained under these conditions.
- In order to express the SNAP-tag or a fusion protein from the provided vector, transform the plasmid into an E. coli expression strain that provides inducible T7-polymerase.
- Pick a colony of the transformed cells the next day and grow a starter culture in liquid medium with selection (e.g. 10 ml of LB containing Ampicillin 100 mg/l) to stationary phase (8 hours or overnight).
- Dilute the starter culture at 1/20 to 1/50 into a suitable volume (e.g. 500 ml) of liquid medium with selection for a main culture. Grow the cells until the OD600 reaches approximately 0.8.
- Induce expression of the T7-polymerase of BL21-AI cells by adding L-arabinose to 0.2% final concentration. This will lead to expression of genes under control of the T7 promoter.
- Shake the culture at 25 to 30°C a further 3 to 8 hours. The yield of soluble protein is increased at this reduced temperature for a number of SNAP-tag fusion proteins.
- Harvest the bacteria by centrifuging the culture for 15 min at 5000 rcf. The bacterial pellets may be stored frozen at -20°C prior to lysis.
- The cells can be lysed and the fusion protein extracted by standard methods.
- The crude lysate can be used directly to perform the SNAP-tag reaction, or the fusion protein can be purified before use.
SNAP-tag fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lysates). The SNAP-tag as provided in the vector does not contain an affinity tag, so it should be purified by standard separation methods of protein chemistry (e.g. ion exchange chromatography, gel filtration). If you have incorporated an affinity tag into your fusion protein, use protocols adapted to that affinity tag.
We recommend the routine addition of 1 mM DTT or other reducing agent (such as b-mercaptoethanol at 1-5 mM) to solutions used for the purification and storage of SNAP-tag fusion proteins. SNAP-tag stability and reactivity in vitro is improved by the presence of a reducing reagent.
The presence of chelating agents such as EDTA should be avoided in solutions used for the expression, purification, and reaction of the SNAP-tag, as the protein contains a structural Zn2+ ion.
Storage and Handling of Unlabeled SNAP-tag Fusion Proteins
Note: Correct storage and handling of unlabeled SNAP-tag fusion proteins is essential to maintain reactivity of the SNAP-tag prior to labeling.
Add 1 mM DTT or other reducing reagent to buffers used for the storage of unlabeled SNAP-tag fusion proteins. Unlabeled protein samples should be stored at -20°C, or at 80°C for long-term storage. Handling at temperatures above 0°C should be minimized by thawing the unlabeled protein samples shortly before use, and keeping them on ice until just before the labeling reaction.
If a particular fusion protein requires buffers without DTT or another reducing agent present, pay particular attention to minimize all handling steps of the protein above 4°C before the labeling reaction itself.
The SNAP-tag itself is tolerant of a wide range of buffers. The requirements of your fusion partner should dictate the buffer selected. From our experience, the following storage buffer composition gives good performance, especially when freezing protein material: pH between 7.0 and 8.0, monovalent salts (e.g. sodium chloride) between 50 mM and 250 mM, at least 1 mM DTT. Non-ionic detergents can be added if required, but ionic detergents should be avoided because they reduce the activity of the SNAP-tag. Many proteins benefit from the addition of glycerol for frozen storage, typically 20% v/v.
Labeling of SNAP-tag Fusion Proteins
The labeling of the fusion protein in solution with SNAP-tag substrates is described in the instructions supplied with SNAP-Cell and SNAP-Surface substrates.
Expression can be analyzed by running samples on an SDS-PAGE gel. For SNAP-tag fusion proteins you should see a band of molecular weight approxi-mately 20 kDa bigger than your protein of interest. SNAP-tag fusion proteins are most easily visualized on a gel using fluorescent imaging after labeling with SNAP-Vista Blue (NEB #S9146) or SNAP Vista Green (NEB #S9147). If no protein can be seen at the expected molecular weight, a Western blot with an antibody against your open reading frame or an affinity tag should give higher sensitivity detection.
In general we have not experienced problems expressing SNAP-tag protein fusions. However if your fusion gene does not appear to be expressed, try expressing the SNAP-tag alone as a positive control, for example using cells transformed with the pSNAP-tag(T7) expression plasmid. You should clearly see a band at approximately 20 kDa in induced cells representing SNAP26b protein. If you do not see expression, check your expression conditions: suitable host strain (BL21-AI™), appropriate selection (ampicillin for pSNAP-tag(T7)), freshly grown cells and correct induction conditions.
If the SNAP-tag alone is expressed but your fusion protein is not, then there are a variety of possible causes:
- It is possible that this fusion protein may be toxic for bacteria. Some rare proteins are ex-tremely toxic when expressed in host bacteria and will not show expression in most E. coli based expression systems. It is difficult to troubleshoot such instances, but the use of a highly repressed, stringently induced promoter may help. Signs of host-cell toxicity could be inhibition of bacterial growth, or even cell lysis, after induction.
- The fusion protein may be unstable in E. coli. There are several reasons why heterologous proteins may be unstable in E. coli such as incomplete folding and rapid protease degradation.
- Some recombinant proteins cannot be expressed in E. coli, in which case use of another expression system should be considered.
Recombinant proteins in E. coli frequently partition partly or totally into inclusion bodies. This may result from over-expression, be caused by limited protein solubility or represent the aggregation and accumulation of improper folding intermediates. SNAP-tag can usually be found in both the soluble and insoluble cell fractions. Generally sufficient SNAP-tag is present in soluble fractions for subsequent purification. Expression of soluble proteins tends to be improved when cells are grown after induction at a reduced temperature such as 25–30°C.