A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli

Abstract

An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL-ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca2+ for 2 days followed by gel filtration, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.

Scheme 1

Procedure for preparation of active recHPL–ST II.

Fig. 1

Purification of recHPL–ST II using Strep-Tactin-Sepharose. (A) Affinity chromatography profile on a Strep-Tactin-Sepharose column. Elution was started from the fraction indicated by the arrow and the peak was obtained at fraction no. 17. (B) SDS-PAGE and western blot analysis of the recHPL–ST II. From left lanes, ‘CBB’, CBB-stained 12% polyacrylamide gel showing MW, marker proteins; Ext, solubilized and refolded fusion protein from inclusion body; Peak, the purified recHPL–ST II on the Strep-Tactin-Sepharose column. ‘Anti-HPL’, the fractions Ext and Peak were electroblotted to a PVDF membrane and stained using anti-lipase antibodies and the colour was developed using ECL as described in the text; ‘Anti-ST II’, the fractions Ext and Peak were electroblotted to a PVDF membrane and stained using anti-ST II antibodies and the colour was developed using ECL as described in the text.

Fig. 2

Effect of additives on the refolding efficiency of recHPL–ST II. Different additives were added into the refolding buffer, namely, 0.5% PEG3400, 1 M glycine, 400 mM l-Arg, 0.75 M sucrose, 10% glycerol and 10% glycerol + 2.5 mM Ca²⁺. Refolding recovery yield was measured relative to the control refolding condition, obtained without additives as standards (n = 3).

Fig. 3

Purification of active recHPL by gel-filtration FPLC. (A) Elution profile of recHPL on gel filtration. RecHPL after refolding was loaded onto a HiPrep 16/60 Sephacryl S-200 column (column volume: 120 ml) and eluted using 25 mM PBS containing 400 mM l-Arg at a flow rate of 0.5 ml/min at 4°C. Specific activity of the fractions was measured using tributyrin as substrate. (B) SDS-PAGE of the fractions eluted from gel filtration chromatography under reducing and non-reducing conditions. Me: 2-mercaptoethanol, Lane S: recHPL after refolding. Lanes numbered as 10–20: correspond to the eluted fractions from No. 10 to 20 in (A), respectively. (C) Specific activity of the recHPL that has the highest activity in the eluted fractions and native PPL using tributyrin or triolein as substrates. Data shown represent the mean ± SD values from at least three measurements.

Fig. 4

The reactivity of the recHPL and PPL against the anti-HPL antibodies by dot blot analysis. (A) Dot blot analysis on PVDF membrane stained with the rabbit anti-HPL IgGs for immunoblotting and the HRP-labelled anti-rabbit IgG Fc. (B) Densitometric analysis of spots on blotting membrane. Data shown represent the mean ± SD.

Fig. 5

Heat stability of the lipolytic activity of recHPL and PPL. Lipolytic activity was measured at the indicated temperatures using the pH-stat technique and tributyrin as substrate. Activities at different temperatures are expressed as a percentage of the activity at 37°C. Data shown represent the mean ± SD values from at least three measurement and were analysed for statistical significance using the Student’s t test. *P < 0.05; **P < 0.01.

Fig. 6

Dependency of the lipolytic activity of recHPL and PPL on pH. Activity was measured using pH-stat technique at the indicated pH value at 37°C. Tributyrin was used as substrate. Activities at different pH are expressed as a percentage of the activity at pH 8.0. 1 mM Tris-HCl and 1 mM phosphate buffer were used as buffers at pH 8.0–9.0 and pH 6.0–7.0, respectively. Data shown represent the mean ± SD values from at least three measurements and were analysed for statistical significance using the Student’s t test.

Fig. 7

Sensitivity of recHPL and PPL to Orlistat. Lipolytic activity was measured at 37°C using the pH-stat technique and tributyrin as the substrate. Activities with different concentrations of Orlistat are expressed as a percentage of the activity. Data shown represent the mean ± SD from at least three measurements.