High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator

Abstract

Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

Figure 1. Constructed plasmid maps of (A) pET-20b(+)-pul, (B) pET-22b(+)-pul, and (C) pET-22b(+)-pulΔlac.

Figure 2. SDS-PAGE analysis of PUL expression in E. coli BL21(DE3)/pET-20b(+)-pul before and after degeneration.

Lane M: protein molecular weight marker; Lane 1: total protein after degeneration; Lane 2: total protein before degeneration.

Figure 3. Analysis of loss and mutation of the recombinant plasmid from E. coli BL21(DE3)/pET-20b(+)-pul.

(A) Restriction enzyme analysis of the recombinant plasmid pET-20b(+)-pul. Lane M: DNA Marker, Lane 1: double digestion of pET-20b-pul; (B) Plasmid stability during cryopreservation; (C) SDS-PAGE analysis of PUL expression in E. coli BL21(DE3)/pET-20b(+)-pul strain. Lane M: protein molecular weight marker, Lane 1: total protein of newly transformed strain, Lane 2: total protein of originally constructed strain.

Figure 4. Effect of cryopreservation time of frozen glycerol stocks on expressed PUL activity in different recombinants.

E. coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul were induced at 20 °C with 0.5 mM IPTG when cell turbidity (OD_{600 nm}) reached 1.2. Fresh transformants were used for expression of the first time, and simultaneously seed culture of the fresh transformants was cryopreserved as frozen glycerol stocks used for the subsequent expressions.

Figure 5. SDS-PAGE analysis of basal expression of PUL in different recombinants.

Lane M: protein molecular weight marker; Lane 1: total protein of E. coli BL21(DE3)/pET22b(+); Lane 2: total protein of E. coli BL21(DE3)/pET22b(+)-pul; Lane 3: total protein of E. coli BL21(DE3)/pET22b(+)-pulΔlac.

Figure 6. Effect of cryopreservation time of frozen glycerol stocks on expressed PUL activity in different recombinants.

E. coli BL21(DE3)/pET-22b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pulΔlac were induced at 20 °C with 0.5 mM IPTG when cell turbidity (OD_{600 nm}) reached 1.2. Fresh transformants were used for expression of the first time, and simultaneously seed culture of the fresh transformants was cryopreserved as frozen glycerol stocks used for the subsequent expressions.

Figure 7. Scheme of double repression involving lac operator regulation for target protein expression.

Figure 8. Expressed PUL activity profiles of different recombinants with auto-induction.

The PUL activities were compared between the two recombinants, E. coli BL21(DE3)/pET-20b(+)-pul (circle) and E. coli BL21(DE3)/pET-22b(+)-pul (square).

Figure 9. SDS-PAGE analysis of PUL expression in different recombinants with auto-induction.

Lane M: protein molecular weight marker; Lane 1: total protein of E. coli BL21(DE3); Lane 2: total protein of E. coli BL21(DE3)/pET20b(+)-pul; Lane 3: total protein of E. coli BL21(DE3)/pET22b(+)-pul.

Figure 10. Effect of glycine concentration on extracellular activity of expressed PUL from E. coli BL21(DE3)/pET22b(+)-pul.

Glycine was added in the auto-induction culture to the final concentration of 0 (star), 0.6% (circle), 0.9% (triangle), and 1.2% (square).

Figure 11. SDS-PAGE analysis of extracellular PUL production from E. coli BL21(DE3)/pET22b(+)-pul with glycine at different concentration.

Lane M: protein molecular weight marker; Lane 1: extracellular fraction secreted without glycine; Lane 2: extracellular fraction secreted with 0.6% glycine; Lane 3: extracellular fraction secreted with 0.9% glycine; Lane 4: extracellular fraction secreted with 1.2% glycine.