Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo

Abstract

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

Figure 1. Schematic map of expression vectors for fusion proteins used in the present study.

A. MBP-TEVP(S219V). B. Trx-rsTEV-_QFAEGFP-6His and bQC(E45Q). C. Trx-rsTEV-MCP1-6His and bQC(E45Q). In B and C, the two restriction enzyme sites designed for the sticky-end PCR cloning strategy are indicated. Abbreviations: MBP, maltose binding protein; TEVP(S219V), a high-stability mutant of tobacco etch virus protease; Trx, thioredoxin; S, S-tag; EGFP, enhanced green fluorescence protein; bQC(E45Q), a gain-of-function mutant of QC from Xanthomonas campestris; MCP1, monocyte chemoattractant protein 1; rsTEV, TEVP recognition site; T₇-P, T₇ promotor; T₇-T, T₇ terminator.

Figure 2. In vivo cleavage and antonomous pGlu formation of EGFP.

A. Schematic representation of induced fusion proteins and cleaved-off products. The theoretical molecular mass of these proteins are indicated. B. SDS-PAGE analysis of the fusion proteins and products. Lane M, molecular markers; lane I, whole-cell lysates of E. coli cells after induction with IPTG; lane N, whole-cell lysates of uninduced cells; lane 1, soluble fraction of the cell lysates; lane 2, unprocessed Trx-rsTEV-EGFP-6His and cleaved-off EGFP-6His purified by a Ni-NTA column; lane 3, EGFP-6His purified by a Superdex-75 size-exclusion column. The possible locations of induced fusion proteins and cleaved-off products are indicated with arrow heads. C. NanoESI-Q/TOF MS analysis of cleaved-off EGFP-6His. Note that a single signal corresponding to _pEFA-EGFP-6His was shown. D. NanoESI-Q/TOF MS analysis of cleaved-off EGFP-6His when the E89A mutant of bQC was used. Note that a strong signal corresponding to the degraded product _PG-EGFP-6His was shown.

Figure 3. NanoLC-MS/MS analysis of cleaved-off products.

A. EGFP-6His. B. MCP1-6His.

Figure 4. In vivo cleavage and antonomous pGlu formation of MCP1.

A. Schematic representation of induced fusion proteins and cleaved-off products. The theoretical molecular mass of these proteins are indicated. B. SDS-PAGE analysis of the fusion proteins and products. Lane M, molecular markers; lane I, whole-cell lysates of E. coli cells after IPTG induction; lane N, whole-cell lysates of uninduced cells; lane 1, soluble fraction of the cell lysates; lane 2, unprocessed Trx-rsTEV-MCP1-6His and cleaved-off MCP1-6His purified by a Ni-NTA column; lane 3, MCP1-6His purified by a Superdex-75 size-exclusion column. The possible locations of induced fusion proteins and cleaved-off products are indicated with arrow heads. C. NanoESI-Q/TOF MS analysis of cleaved-off MCP1-6His. Note that a strong signal corresponding to _pE-MCP1-6His was shown.

Figure 5. Cell migration activity of purified MCP1-6His.

The cell migration activities of MCP-6His at varied concentrations, as indicated, were evaluated by using U937 cells as described in the Materials and Methods. The CI values, shown as mean ± SEM, were calculated from five independent experiments. ***P<0.001.