Development of T7 expression system for high-level expression of mTGase in C. glutamicum
To enhance the expression of mTGase, a T7 RNA polymerase (RNAP) -dependent expression system was developed for C. glutamicum [35]. The lac promoter in the plasmid pXMJ19 was replaced by the T7 promoter, yielding the plasmid pXMJ19T7. Correspondingly, the gene fragment containing the T7 RNAP and lacI gene was integrated into the chromosome of C. glutamicum, yielding the strain C. glutamicum ATCC 13032 (DE3). All the C. glutamicum strains used in the following experiment were C. glutamicum ATCC 13032 (DE3) unless otherwise specified. To test the T7 expression system, the gene encoding pro-mTGase was cloned on the pXMJ19T7 and expressed under the control of the T7 promoter (Fig. 1A). As shown in Fig. 2A, a strong band at approximate 43.3 kDa was observed in cell lysate, suggesting the high-level expression of pro-mTGase in C. glutamicum. Furthermore, to secret pro-mTGase, the cspA signal peptide was fused to pro-mTGase and the resulting fusion protein was expressed by using this T7 expression system (Fig. 1B). A strong band corresponding to the 43.3 kDa pro-mTGase was observed in the culture supernatant, suggesting that the high-level expression and secretion of pro-mTGase (Fig. 2B). Meanwhile, a clear band corresponding to 45.7 kDa was observed in the cell lysate, indicating that some of the fusion protein was not secreted but resided intracellularly. All these results demonstrated that the T7 RNAP-dependent expression system was successfully established in C. glutamicum and can be used in the following study.
Intein Ssp DnaB was subjected to the premature cleavage in C. glutamicum
Despite pro-mTGase was expressed intracellularly and extracellularly at a high level by using the T7 expression system, no obvious enzyme activity was detected for pro-mTGase, which needs to be activated. To activate pro-mTGase, intein Ssp DnaB was fused with the pro-mTGase. The Ssp DnaB gene was inserted between gene fragments encoding pro-peptide and mature part of mTGase. The chimeric gene was cloned on pXMJ19T7 and expressed under the control of the T7 promoter, yielding the plasmid pXMT7-csp-pro-ssp-mTG (Fig. 1C). The expression of the fusion protein was then analyzed by SDS-PAGE. No clear band corresponding to fusion protein (60.7 kDa) or mature mTGase (38.8 kDa) was observed in the culture supernatant. Meanwhile, no mTGase activity was detected in the culture supernatant. Moreover, no mTGase activity was detected after the culture supernatant was treated under pH 6.5 for 24 hours, which was supposed to induce the cleavage. These results indicated that the precursor fusion protein was not secreted to the culture. However, in the cell lysate supernatant, a specific band that exactly matched the molecular weight of the mature part of mTGase (38.8 kDa) was observed (Fig. 3A). Meanwhile, the mTGase activity (0.2 U/mL/OD) was also detected in the cell lysate, further indicating the intracellular production of active mTGase. These results suggested that the intein Ssp DnaB was subjected to the premature in vivo cleavage when it was applied to activate the pro-mTGase. Consequently, the premature cleavage activated the pro-mTGase intracellularly and prevented the pro-mTGase secretion.
Premature cleavage was applied to active mTGase intracellularly
Observing the production of active mTGase through premature cleavage, we employed rather than suppressed premature cleavage to produce active mTGase intracellularly in C. glutamicum. To express the mTGase precursor intracellularly, the gene fragment encoding cspA signal peptide was removed from the plasmid pXMT7-csp-pro-ssp-mTG and the resulting plasmid was named pXMT7-pro-ssp-mTG (Fig. 1D). As shown in Fig. 3A, the active mTGase was successfully produced intracellularly in C. glutamicum. The removal of the cspA signal peptide increased the intracellular expression level of the active mTGase. Meanwhile, the mTGase activity of the cell lysate supernatant reached to 0.7 U/mL/OD, which is increased by 2.5 fold. To observe the process of premature cleavage, the expression of the fusion protein pro-ssp-mTG was analyzed at different time intervals. The protein bands for fusion protein continued to increase in 8 hours and began to decrease after 8 hours. Meanwhile, the protein bands for mature mTGase was observed in 4 hours and increased constantly thereafter (Fig. 3B). This result indicated that the premature cleavage of intein Ssp DnaB efficiently activated the pro-mTGase intracellularly in C. glutamicum. Furthermore, the substitution of the first C-extein residue with proline was presumed to inhibit the self-cleaving activity of Ssp DnaB [36]. As we expected, the proline substitution resulted in the accumulation of precursor fusion protein. Meanwhile, the production of mature mTGase was significantly decreased (Fig. 3C). This result further indicated the premature cleavage of Ssp DnaB mediated the cleavage of the precursor fusion protein, resulting in the intracellular production of active mTGase.
Besides, the recombinant mTGase with the His6 tag at the C-terminus was purified from the supernatant of cell lysate and the N-terminal amino acid sequencing was conducted. Five residues M-D-S-D-D, which were exactly matched with the mature mTGase N-terminus, were identified by the N-terminal sequencing. This result strongly suggested that the premature cleavage of Ssp DnaB activated the mTGase in a highly specific manner.
The first C-extein residue modulated the premature in vivo cleavage
The first C-extein residue is of vital importance to the modulation of C-terminal cleavage [36]. Thus, the Met in the +1 position of C-extein was substituted with the other 19 naturally occurring amino acids. The premature cleavage efficiency of each variant of mTGase was compared by analyzing the intracellular mTGase activities. As shown in Fig. 4, the mTGase variant with methionine in the +1 position exhibited the highest mTGase activity. Compared to the variant with Met at the +1 position, the variant with Leu at +1 position exhibited 78% activity, suggesting the Leu was the next favored residue for C-terminal cleavage. Fifteen variants with substitutions (Val, Phe, Ser, Ile, Trp, Arg, Asn, Gly, Ala, Pro, Glu, Tyr, Gln, Cys and Thr) yielded various extent of C-terminal cleavage (10–65%), while three substitutions (Asp, His and Lys) essentially decreased the cleavage efficiency to less than 10%. These results demonstrated that the substitution of the first C-extein residue modulated the premature cleavage and the resulting intracellular production of mature mTGase. The variant with methionine residue at the +1 position of the C-extein exhibited the highest intracellular mTGase activity and hence was used in the following experiment.
Characterization of recombinant mTGase
As shown in Fig. 5A and Fig. 5B, the optimal temperature and pH of the recombinant mTGase was 55 °C and 7.0, respectively. Furthermore, the enzyme was stable at 40 °C, and 30% of the activity was retained at 50 °C for 100 minutes. However, at 60 °C, the recombinant mTGase lost all its activity within 20 minutes (Fig. 5C). In addition, the purified recombinant mTGase was stable at pH 5.0–9.0 after 1 h incubation at room temperature, during which more than 70% activity was retained (Fig. 5D). The effects of inhibitors and various metal ions on the activity of recombinant mTGase were also detected. It was found that the activity of purified mTGase was not inhibited by Ethylene Diamine Tetraacetie Acid (EDTA), phenyl methyl sulfonyl fluoride (PMSF) and metal ions including Na+, K+, Mg2+ and Ca2+. The enzyme activity was mildly inhibited by metal ions, such as Cu2+, Mn2+, Fe2+. With the addition of Zn2+, the activity was almost totally inhibited (Table 1). These results demonstrated that the major properties of the recombinant mTGase produced in this study were not noticeably altered [20].
High-level production of active mTGase in a jar fermentor
The recombinant mTGase production was scaled up by using a 1 L fed culture. Cells were grown at 30 °C and then changed to 25 °C after IPTG was added. Samples were taken at different time intervals and the enzyme activity was determined. As shown in Fig. 6, the mTGase activity was constantly detected after IPTG induction owing to the premature cleavage. At 42 h, the mTGase activity was up to 49 U/mL, which was the highest intracellular mTGase activity ever reported. It is highly likely that a higher level production of mTGase would be available once the fermentation process is further optimized.