Ligation independent cloning vectors for expression of SUMO fusions

Abstract

With demand increasing for the production of many different proteins for biophysical or biochemical analyses, rapid methods are needed for the cloning, expression and purification of native recombinant proteins. In particular, generic methods are required that are independent of the target gene sequence. To address this challenge we have constructed four Escherichia coli expression vectors that can be used for ligation independent cloning (LIC) of an amplified target gene sequence. These vectors represent the combinatorial pairing of two different parent vector backbones with two different affinity tags. The target gene is cloned downstream of the sequence coding for an affinity-tagged small ubiquitin related modifier (SUMO). Using enhanced green fluorescent protein (eGFP) as an example we demonstrate that the LIC procedure works with high efficiency for all four of the vectors. We also show that the resultant recombinant SUMO fusion proteins can be overexpressed in E. coli and readily isolated by standard affinity purification techniques. Importantly, the purified fusion product can be treated with recombinant SUMO hydrolase to yield a mature target protein with any residue except proline at the amino terminus. We demonstrate an application of this by generating recombinant eGFP containing a non-native amino terminal cysteine residue and using it as a substrate for expressed protein ligation (EPL). The reagents and techniques described here represent a generic method for the rapid cloning and production of a target protein, and would be appropriate for a high throughput genomic scale expression project.

Figure 1

Map and details of the cloning sites for the LIC-SUMO plasmids. (A) Map of pET and pASK based plasmids showing the topology of the encoded sequences (B) The sequence of the common cloning site found in the pET- and pASK-based vectors. Two alternative affinity tags are available for each plasmid backbone, either a hexa-histidine (His₆) or a Strep-II sequence (boxed region). The tags are encoded in the same reading frame with the amino terminus of the yeast SUMO sequence. Cleavage of fusions with the Sumo-specific protease (dtUD1) occurs on the carboxyl side of the terminal glycine of the Sumo sequence, at the position marked by the arrow.

Figure 2

Flowchart describing the method used to generate long single stranded overhangs for LIC. (A) Preparation of the plasmid. Each plasmid is digested with BseRI and then treated with calf intestine alkaline phosphatase (CIAP) to minimize self ligation. Following restriction digestion the resulting product can be directly treated with T4 DNA polymerase in the presence of dCTP to generate the single stranded overhangs. Alternatively the digested product can first be amplified by PCR and then treated with T4 DNA polymerase; the PCR will yield a blunt-ended product lacking the underlined bases. (B) Preparation of the insert. The target sequence is amplified by PCR using primers containing the underlined 5' extensions. The resultant product is treated with T4 DNA polymerase in the presence of dGTP to generate single stranded ends complementary to those in the plasmid. As the forward primer extension encodes the C-terminal residues of SUMO (shown above sequence), the final base of which terminates the T4 DNA polymerase exonuclease activity in the presence of dGTP, the first codon (nnn) of the amplified target can encode any residue.

Figure 3

12% SDS-PAGE Coomassie stained gels showing subtractive purification of AtxQ30. Lane 1, Cell lysate; Lane 2, Supernatant following clarification by centrifugation; Lane 3, StrepTactin column flowthrough; Lane 4, desthiobiotin elution; Lane 5, dtUD1 catalysed cleavage reaction; Lane 6, Dialysed cleavage reaction; Lane 7, Subtracted flowthrough from StrepTactin column; Lane 8, Desthiobiotin elution following second pass through the StrepTactin column. Lanes 4 to 8 are loaded at twice the relative concentration of the cell lysate sample.

Figure 4

10-20% SDS-PAGE Coomassie stained gels showing subtractive purification and expressed protein ligation (EPL) of eGFP. Position of molecular weight markers are shown (kDa units). (A) Subtractive purification of His₆SUMO fusion of eGFP. Lane 1, Cleared cell lysate; Lane 2, Nickel column flowthrough; Lane 3, 7.5mM imidazole wash; Lane 4, 250 mM imidazole elution; Lane 5, dtUD1 catalysed cleavage reaction; Lane 6, 250 mM Imidazole elution following second nickel column pass; Lane 7, Subtracted flowthrough from nickel column. (B) Ligation of eGFP containing an amino terminal free cysteine to MBP. Lane 1, Purified MBP-thioester; Lane 2, Purified eGFP; Lane 3, EPL reaction 0 hrs; Lane 4, EPL reaction 24 hrs.