Expression of double-tagged GlcNAc 2-epimerase
A 1209 bp PCR fragment encoding the Synechocystis sp. PCC6803 GlcNAc 2-epimerase gene, plus a sequence encoding 5D, was cloned into pGEX-2TK between the BamHI and EcoRI sites. The resulting plasmid contained a in-frame fusion of GST, GlcNAc 2-epimerase and 5D, and was confirmed by DNA sequencing. Under the inducing conditions indicated in Methods, E. coli BL21 harboring the gene fusion produced a significant level of double-tagged GlcNAc 2-epimerase when grown to an OD600 of approximately 1.0 and treated with IPTG to induce gene expression. As shown in Figure 1a, the expression level was nearly independent of IPTG concentration between 0.01 to 1 mM, when induction was for 7 h. The fusion protein was largely insoluble (dark diffusive band in lane P for all cases). At every IPTG concentration, only a low level of fusion protein was seen in the supernatant fractions (S) of crude protein extracts.
The expression level of fusion protein increased significantly with induction time, as shown in Figure 1b–1d. One hour after induction, the protein became evident in the S fraction, increased as induction time reached to 3 h, and remained somewhat constant through 5–7 h of induction. In contrast, protein in the P fraction dramatically increased after 3 h, and increased proportionally with time of induction afterward. The specific GlcNAc 2-epimerase activity of protein in the S fraction increased slightly after 3 h of induction, to about 0.05 U/mg when measured as the conversion of ManNAc to GlcNAc, and remained unchanged thereafter. P fractions containing the overexpressed proteins had relatively higher activities. As shown in Figure 1b, the specific activity increased by approximately 2.5 fold in the P fraction after 3 h induction, and after 7 h, the specific activity had risen to 0.32 U/mg, corresponding to approximately 6.5 times the activity of the soluble fractions. These data suggest that the proteins in the precipitate were active, and formed aggregates because their concentrations were much higher than the protein solubility. P fraction samples were prepared by re-dissolving the precipitate of the crude protein extract with 5 ml deionized water. The tremendous increase in specific activity in the P fraction was mainly due to the enrichment of overexpressed double-tagged protein in the precipitated form.
Using the Compute pI/Mw tool in the ExPASy Proteomics Server http://www.expasy.org/, the theoretical molecular mass for GST-GlcNAc 2-epimerase-5D was predicted to be 73.2 kDa. The introduction of the GST tag allows the double-tagged fusion protein to be purified from the protein extract using conventional affinity methods. After GSH-affinity purification, the purified GST-GlcNAc 2-epimerase-5D had a specific activity of 11.5 U/mg protein using ManNAc as the substrate. Previous studies on GlcNAc 2-epimerase indicated that this enzyme has a pH optimum of 8 [20], with high activity at pH 7–8, but no activity below pH 6. Since the theoretical pI for native GlcNAc 2-epimerase is 5.59, this means the enzyme is likely to be active only in deprotonized conditions, when pH>pI. The addition of GST to generate GST-GlcNAc 2-epimerase, including the linker sequences, led to a theoretical increase in pI to 5.89. We thus designed a 5D tag at the C-terminus to bring the theoretical pI back to 5.59.
The introduction of tags not only increased the protein solubility, but also altered the thermal stability. A previous study showed that the optimal temperature for GlcNAc 2-epimerase from Synechocystis sp. PCC6803 is 37°C [11]. The double-tagged fusion protein was determined to have an optimal temperature of 50°C, suggesting a higher operation temperature was possible for the application of double-tagged GlcNAc 2-epimerase to sialic acid production. The Michaelis constant, K
m
, for purified GST-GlcNAc 2-epimerase was determined to be 7.7 mM using ManNAc as substrate. This value was slightly higher than the 4.76 mM determined earlier [11] for native GlcNAc 2-epimerase from the same source. Pyruvate is a competitive inhibitor for this enzyme. The inhibition constant of pyruvate for GST-GlcNAc 2-epimerase-5D was 31 mM, which was very close to the 36 mM determined for single-tagged protein GST-GlcNAc 2-epimerase [20].
Expression of double-tagged Neu5Ac aldolase
Sequences (909 bp) encoding Neu5Ac aldolase and 5R were amplified from E. coli. The PCR product was purified and cloned into pGEM-T Easy Vector, before excision and insertion into pGEX-2TK between the BamHI and EcoRI sites. This construction created a fusion of Neu5Ac aldolase tagged with GST at the N-terminus and 5R at the C-terminus, with the gene for the double-tagged protein under the regulation of the tac promoter. The fusion protein contains a total of 535 amino acid residues comprising Neu5Ac aldolase, the two tags, and the sequence linking GST and Neu5Ac aldolase. Non-tagged Neu5Ac aldolase contains 297 amino acid residues and has a computed MW of 32.6 kDa and theoretical pI value of 5.61, while the computed MW and pI for the fusion protein are 60.4 kDa and 6.84.
Similar to GST-GlcNAc 2-epimerase-5D, GST-Neu5Ac aldolase-5R could also be overexpressed in E. coli. SDS-PAGE results for GST-Neu5Ac aldolase-5R (Figure 2a) were similar to those for GST-GlcNAc 2-epimerase-5D. Unlike the induction of GST-GlcNAc 2-epimerase-5D, however, the expression level of GST-Neu5Ac aldolase-5R depended on inducer concentration. The use of 0.01 M IPTG led to a significantly lower level of protein expression than 0.1 and 1 mM. The fusion protein was largely insoluble (dark diffusive band in lane P). The band showing the level of soluble protein (lane S) was indistinct, suggesting a relatively low solubility for GST-Neu5Ac aldolase-5R.
Overexpression of GST-Neu5Ac aldolase-5R was confirmed by analyzing the purified fusion protein after affinity purification. Figure 2b and 2c show the SDS-PAGE analysis and Western blotting of GST-Neu5Ac aldolase-5R from GSH-affinity column fractions. Western blotting using antibody against the GST tag showed that the molecular mass of double-tagged protein was close to the theoretical value of 60.4 kDa.
In the fused construct in plasmid pGEX-2TK, a thrombin recognition site lies between GST and Neu5Ac aldolase, so the GST tag could be released from the fusion protein by thrombin digestion. Cleavage of the double-tagged fusion protein by thrombin resulted in two proteins, GST and Neu5Ac aldolase-5R. Both the double-tagged fusion protein GST-Neu5Ac aldolase-5R, and the GST-released fusion protein Neu5Ac aldolase-5R were able to bind SP Sepharose. The typical adsorption curve displayed a change in adsorbed fusion protein with time. A sharp decrease of proteins in solution was observed within the first few hours, followed by a very slow decrease during the immobilization process. This was seen using two preparations of the purified fusion proteins. Although the theoretical pI values for GST-Neu5Ac aldolase-5R and Neu5Ac aldolase-5R with their linker sequences are 6.84 and 8.27, the experimental pI values were estimated as 8.3 and 8.8 from two-dimensional gel electrophoresis. Thus, they could easily be immobilized on the cationic exchanger SP Sepharose.
Immobilization of fused proteins
For the production of sialic acid, Neu5Ac, using GlcNAc 2-epimerase and Neu5Ac aldolase in a single pot, both enzymes should be active at the operational conditions, including pH and temperature. A pH in the range of 7–7.5 should be optimal, because in that range, the double-tagged Neu5Ac aldolase would be positively charged, since its pI value was increased by the introduced 5R tag. This allowed the fusion protein to be easily adsorbed onto cationic SP Sepharose resins. In contrast, the double-tagged GlcNAc 2-epimerase would be negatively charged and able to bind to the anionic exchanger Q Sepharose. Immobilization of the GST affinity-purified fusion proteins onto ion exchange resins was achieved in a few hours. Greater concentrations of proteins mixed with the ion exchanger caused a longer adsorption time before adsorption equilibrium was reached. If a ten-hour adsorption was used, the amount of fusion protein in the incubated solution could be increased, leading to more adsorbed protein on the resin (Figure 3). When the applied concentration was 0.2 mg/ml, about 90% of the purified fusion protein could be immobilized. When the applied concentration was 2.2 mg/ml, the immobilized protein approached 5.6 mg protein/g resin, corresponding to immobilization of approximately 30% of the proteins. Immobilization led to a reduction in enzymatic activity of about 25%, based on the specific activity of purified protein as 100%.
Time courses of immobilization of double-tagged GlcNAc 2-epimerase on Q Sepharose were similar to those for immobilization of double-tagged Neu5Ac aldolase on SP Sepharose. A similar pattern (Figure 3) was seen for the influence of the applied concentration of purified GST-GlcNAc 2-epimerase-5D on the amount of protein bound to Q Sepharose.
Since the lysis of cells overexpressing the tagged protein resulted in a large amount of enzymatically active protein in the precipitate, repeated lysis (typically three times) was employed to increase the volume of lysis buffer as well as the extent of cell disruption and protein dissolution. All supernatants were pooled and used as the crude protein extract for fusion protein immobilization. By using the ionic tags, direct capture of fusion protein from the crude protein extract was very effective. As shown in Figure 4, the amount of protein immobilized on the resins increased with the loaded concentration of protein. For double-tagged Neu5Ac aldolase binding to SP Sepharose, the amount of bound protein approached 5.0 mg protein/g-resin when the crude extract protein concentration was 1.2 mg/ml. The bound protein concentration remained at this level even if the applied concentration was higher, suggesting saturation of protein adsorption. Like the adsorption of purified double-tagged Neu5Ac aldolase to the same ion exchange resin, the fraction of crude protein adsorption decreased with increased protein loading concentration. If the loaded concentration of protein was 2.1 mg/ml, only 30% of proteins in the crude extract were immobilized on the resin. A purification effect was seen for selective immobilization of fusion proteins through their ionic interactions. When a crude, 1.1 mg/ml double-tagged Neu5Ac aldolase solution with a specific activity of 0.75 U/mg was applied to SP Sepharose, the protein eluted from the ion exchanger with 1 N NaCl possessed a specific activity of 2.5 U/mg.
Similar adsorption behavior was observed for the immobilization of GST-GlcNAc 2-epimerase-5D on Q Sepharose. When the loading concentration of crude protein was 2.2 mg/ml, the saturated adsorbed density of protein was 5.3 mg protein/g resin. Under these conditions, about 30% of proteins from the crude extract were immobilized on the resin. Since the protein was not purified prior to immobilization, the specific activity of the immobilized fusion protein was lower than that of immobilized, purified fusion protein. Based on the activity of immobilized, purified GST-Neu5Ac aldolase-5R, the specific activity of immobilized GST-Neu5Ac aldolase-5R via direct capture from crude extract was only 21%. The specific activity of immobilized GST-GlcNAc 2-epimerase-5D via direct capture from crude extract was 37% of the activity of immobilized, purified GST-GlcNAc 2-epimerase-5D. However, the specific activity of immobilized protein obtained by direct capture from crude extract was still much higher than that of protein from crude extract, suggesting a purification effect from selective immobilization through the ionic interaction. When a 1.1. mg/ml crude GST-GlcNAc 2-epimerase-5D solution with a specific activity of 1.6 U/mg, using GlcNAc as the substrate, was applied to Q Sepharose, protein from the ion exchanger with 1 N NaCl possessed a specific activity of 3.4 U/mg.
Production of Neu5Ac using two immobilized double-tagged fusion proteins
Both the soluble and immobilized forms of double-tagged 2-epimerase-5D were effective for the epimerization of GlcNAc and ManNAc. Using GlcNAc as the substrate, the conversion rate catalyzed by purified GST-GlcNAc 2-epimerase-5D increased with the dose of fusion protein and the reaction time. The double-tagged Neu5Ac aldolase immobilized on the cationic exchanger SP Sepharose was able to catalyze the formation of KDN from D-mannose and pyruvate [21].
Figure 5 shows a typical, small-scale run of Neu5Ac production using GST-GlcNAc 2-epimerase-5D immobilized on Q Sepharose (3 g, corresponding to 2430 U/l) and GST-Neu5Ac aldolase-5R immobilized on SP Sepharose (15 g, corresponding to 7161 U/l) as the biocatalysts. Since pyruvate is a substrate for the second reaction but an inhibitor for enzyme of the first reaction, its concentration must be kept below 100 mM, although not too low, during the reaction. Pyruvate was thus added to the reaction whenever its concentration went below 50 mM. Furthermore, a low temperature was chosen to favor equilibrium to Neu5Ac, although this resulted in a slower reaction rate. Shifting the temperature from 30 to 20°C in the middle of the coupling reactions might lead to the gain of high conversion yield, as suggested in the literature [4]. Finally, a conversion of about 68% based on the production of Neu5Ac from GlcNAc on a molecular basis was achieved in 80 h, corresponding to a volumetric productivity of 0.52 g Neu5Ac l-1 h-1. If the reaction was stopped at 56 h, conversion was 62%, corresponding to a productivity of 0.69 g Neu5Ac l-1 h-1. An experimental run using 2000 U/l GlcNAc 2-epimerase and 8000 U/l Neu5Ac aldolase on concentrated substrates 816 mM GlcNAc and 483 mM pyruvate at 30°C, resulted in a conversion of 68% at 140 h, corresponding to a productivity of 0.92 g Neu5Ac l-1 h-1. A final conversion of 77% was seen after 240 h of reaction, corresponding to a productivity of 0.60 g Neu5Ac l-1 h-1) [3]. Our results suggest that the coupled enzymatic system using immobilized double-tagged proteins has practical potential. In the present study, however, the ratio of pyruvate to GlcNAc in the late phase of reaction was kept at a higher level than described previously [3]. Further experiments need to be carried out to avoid using a large excess of pyruvate.
In another Neu5Ac synthesis reaction, combined doses of 1850 U/l GST-GlcNAc 2-epimerase-5D immobilized on Q Sepharose and 1682 U/l GST-Neu5Ac aldolase-5R immobilized on SP Sepharose were used as biocatalysts. A conversion of 35% could be achieved in 28 h, corresponding to a productivity of 0.77 g Neu5Ac l-1 h-1 at 30°C, on the same initial concentration of substrates.