Page 39 - Macromolecular Crystallography

28 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 2.2 In-Fusion™
1. PCR products with approximately 15 bp extensions at 5. Plate on 24-well format LB agar supplemented with
each end that are homologous to the ends of the linearized antibiotic and, if appropriate, X-Gal and IPTG (dilute a 20%
vector should be gel purified for best results. To obtain the X-Gal, in dimethyl formamide, stock 1:1000, dilute the IPTG
best results with lowest non-recombinant ‘background’ 500 mM stock 1:500 in warm agar before pouring). Plate
the linearized vectors should also be gel purified. Take 10 µl of cells, shake plates well to spread the cell
10–100 ng of insert and 100 ng vector in a total volume suspension, and allow at least 10–15 min for the plates
of 10 µl of either 10 mM Tris, pH 8.0 or sterile H 2 O. to dry off before inverting.
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2. Add this to a well of the dry-down In-Fusion™ plate. 6. Incubate overnight at 37 C.
Mix contents briefly by pipetting up and down. 7. Pick colonies for plasmid miniprepping as usual (if
3. React for 30 min at 42 C. blue–white screening is used then blue colonies should
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4. Dilute IMMEDIATELY with 40 µl TE Buffer (10 mM Tris, constitute 10% if the reactions were successful. (The
pH 8.0, 1 mM EDTA) and either transform IMMEDIATELY or blue colonies are derived from inefficiently linearized
freeze the reaction until you are ready to transform. E. coli parental plasmid and should not be picked as they are
8
with a transformation efficiency in excess of 1 × 10 are non-‘recombinant’). Two colonies are normally sufficient
recommended and if the vector has been modified for to find a recombinant clone but more may be required.
blue–white screening ensure that an appropriate E. coli 8. PCR screen colonies or plasmid minipreps as usual with
host strain is used; 5 µl of the diluted reaction should give a vector-specific (e.g. T7) forward primer and your
tens to hundreds of colonies per well of a 24-well plate. gene-specific reverse primer.

2.2.2.2 Tags The use of a short fusion tag, typically seven to
Several fusion protein vectors have been developed eight amino acids, does not necessitate introduc-
for recombinant protein expression since it is recog- tion of a cleavage site to remove the tag. However,
nized that fusion vectors can enhance productivity His-tagged vectors with cleavage sites are available.
and/or solubility of target proteins compared to Combining fusion proteins with N-His tags offers
non-fusion versions. Typically, the fusion partner the potential benefits of improved expression levels
resident in the expression vector is joined to the and solubility of the fusion partner with a generic
N-terminus of the protein of interest via a linker purification strategy.
region containing a protease cleavage site to enable Thereisnoclearconsensusastowhichfusionpart-
subsequent removal of the fusion protein. The most ners give the best performance in terms of enhancing
commonly used proteases are Tev (Parks et al., 1994) expression and solubility, though fusion proteins
and 3C (Cordingley et al., 1989) since both have generally perform better than short His tags (Braun
highly specific linear recognition sequences that are et al., 2002; Dyson et al., 2004; Hammarstrom et al.,
very rarely encountered in other sequences, thereby 2002). However, cleaving off the fusion partner
minimizing the risk of cleavage within the target can lead to precipitation of the target protein. Due
protein (see Section 2.4 for experimental details). to these uncertainties, most structural proteomics
Some of the most commonly used tags also pro- projects have opted to work exclusively with His-
vide affinity purification strategies, for example tagged proteins and invest effort into selecting the
glutathione S-transferase (GST) binds to immobi- targets in terms of domain definition (Folkers et al.,
lized glutathione and maltose binding protein binds 2004). Recently, fusion vectors which combine many
to amylose matrices enabling selection of the fusion of the above attributes have been developed using
protein (Smith and Johnson, 1988; Alexandrov et al., ubiquitin-like proteins (Ubls) as the fusion partner
2001). As an alternative to expressing proteins as (Baker, 1996). Ubls (e.g. SUMO) are small (approxi-
relatively large hybrids, short N- or C-terminal hex- mately 100 amino acid), eukaryotic proteins that are
ahistidine tags are used to facilitate purification known to exert chaperone-like effects on fused pro-
by metal chelate chromatography (see Section 2.4). teinsin E.coli andyeast(Butt etal., 1989). Purification