Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Abstract

Background: Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.

Results: The histidine tag-based self-cleavage method for purifying proteins produced by the wheat cell-free protein synthesis system showed high background, low recovery, and unexpected cleavage between the N-terminally fused sortase and target protein during the protein synthesis. Addition of calcium chelator BAPTA to the cell-free reaction inhibited the cleavage. In order to adapt the sortase-based purification method to the cell-free system, we next used biotin as the affinity tag. The biotinylated sortase self-cleavage purification (BISOP) method provided tag-free, highly purified proteins due to improved recovery of proteins from the resin. The N-terminal sequence analysis of the GFP produced by the BISOP method revealed that the cleavage indeed occurred at the right cleavage site. Using this method, we also successfully purified the E2 heterocomplex of USE2N and USE2v1. The c-terminal src kinase (CSK) obtained by the BISOP method showed high activity in phosphorylating the Src protein. Furthermore, we demonstrated that this method is suitable for automatically synthesizing and purifying proteins using robots.

Conclusion: We demonstrated that the newly developed BISOP method is very useful for obtaining high quality, tag-free recombinant proteins, produced using the cell-free system, for biochemical and structural analyses.

Figure 1

Synthesis of srtA-fusion proteins using the wheat germ cell-free system. A. Schematic representation of the pEU-His-srtA-LPETG-Gene plasmids created using the Gateway system. B. Autoradiogram of SDS-PAGE of proteins synthesized using the cell-free system in the presence of [¹⁴C] Leu. Lane M, Protein MW standards labeled by using [¹⁴C]-containing felt pen. C. Autoradiogram of [¹⁴C] Leu incorporated GFP and SGK495 proteins synthesized by the wheat cell-free system in the presence of the Ca²+ chelating reagent BAPTA. The number represents concentration (mM) of BAPTA used in the protein synthesis reaction. Arrowheads denote the sizes of the full-length proteins. D. Rate of synthesis of the full-length protein and productivity of GFP (pink-colored bar and red-colored line) and SGK495 (purple-colored bar and blue-colored line) in the presence of different concentrations of BAPTA. E. Autoradiogram of [¹⁴C]-Leu incorporated proteins synthesized by the cell-free system in the presence of BAPTA. Asterisk denotes the sizes of the full-length proteins. F. Rate of synthesis of the full-length protein and productivity of proteins in the presence of different concentrations of BAPTA. Productivities of total synthesized and full-length proteins indicated as blue and red bars respectively. G. Purifications of proteins by the cell-free synthesis using the pEU-His-srtA-LPETG-Gene plasmid constructs. CBB-stained protein bands on the SDS-PAGE gel of the eluted (left panel) and resin-bound (right) target proteins are indicated using asterisk. Arrow represents the cleaved His-tagged srtA. Lane M (both panels): Protein MW standards.

Figure 2

Preparation of proteins by the biotinylated-sortase self-cleavage purification (BISOP) method. A. Scheme for creating the pEU-BISOP-LPETG-gene plasmids using the Gateway recombination method. The LPETG-fused target gene containing entry clone plasmid was recombined with the pEU-BISOP-GW vector by using the Gateway LR reaction. B. CBB-stained SDS-PAGE comparing the His- or biotin-tagged GFP protein (indicated using an asterisk) purified manually or by using an automated robot. Lane M: Protein MW standards. C. Biotinylation of GFP, Pfs25 (S25) and CSK proteins synthesized by the cell-free system with vectors having a single (S) or a double (D) biotin ligation site. Biotinylated proteins were detected by labeling the separated protein bands with streaptavidin-Alexa488 as described in the text, followed by scanning using the Typhoon Imager. D. CBB-stained eluted (left panel) and resin-bound (right panel) proteins. Arrow indicates the cleaved biotinylated srtA protease left on the resin. Samples (5 or 10 μL in left or right panel respectively) were loaded on the gel. E. Left panel: Biotinylated proteins were detected by streaptavidin-Alexa488; Right panel: CBB-stained proteins purified by the BISOP method. Samples (5 μL) were loaded on the gel. F. Amino acid sequences from the N-terminal end of GFP purified by the BISOP method were determined by using an amino acid sequencer (asterisks, upper panel). Rounds indicate the number of Edman degradation cycle. Underlined amino acid sequence in the lower panel shows the determined sequence. Arrowhead indicates the site cleaved by the sortase enzyme.

Figure 3

Application of BISOP method to purify the UBE2N/UBE2v1 heterocomplex co-expressed by the cell-free system. A. The proteins were synthesized from the plasmid constructs using the cell-free system in the presence of ¹⁴C-Leu and the synthesized proteins were detected by autoradiography after electrophoresis on a SDS-PAGE (5-20% gradient gel). Lane 1: Proteins synthesized from a mixture of pEU-BISOP-LPETG-UBE2N and pEU-UBE2v1 plasmids; Lane 2: Protein synthesized using the pEU-BISOP-LPETG-UBE2N plasmid; Lane 3: Protein synthesized using the pEU-UBE2v1 plasmid; Lane 4: Sortase cleavage of lane 1 by the BISOP method; Lane 5: Sortase cleavage of lane 2 by the method; and Lane M, Protein MW standards. Asterisk, arrow and arrowhead indicate srtA-LPETG-UBE2N, G-UBE2N and UBE2v1 (tag-free), respectively. B. BISOP was performed after expression of biotinylated srtA-LPETG-UBE2N with (lane 1) or without UBE2v1 (lane 2). The eluted proteins were separated on the SDS-PAGE gel and then stained with CBB. Arrow and arrowhead indicate the purified G-UBE2N and UBE2v1, respectively. C. Scheme for the preparation of UBE2N/UBE2v1 heterocomplex by BISOP.

Figure 4

Activity of CSK prepared using the BISOP method. Biotinylated Src protein was incubated with UBE2N, CSK purified using the BISOP method (eluted CSK and UBE2N), or commercially available GST-CSK fusion protein in the presence of cold (for immunoblot) or radioisotope-labeled (for autoradiogram) ATP. Src phosphorylation was assayed by immunoblot analysis using the anti-Phospho-Src (Y530) antibody. Autoradiogram shows phosphorylation of the Src protein by CSK.