Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: the turkey

Abstract

Background: The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme.

Results: The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined.

Conclusions: Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.

Figure 1

Analysis by electrophoresis performed on 1% agarose gel of PCR products. Lane 1: 1 kb Ladder; lane 2: amplified products obtained using pGAPZαA vector as template; Lane 3, 4, 5, 6,7 and 8: amplified products obtained using as template the genomic DNA extracted from clones transformed with the vector pGAPZαA/TPL (C1, C2, C3, C4, C5, C6).

Figure 2

Time-course of rTPL expression. (A) The six isolated clones of recombinant P.pastoris. (B, insert) The time-course of the rTPL expression by the selected clone (C5).

Figure 3

Q-PCR analysis of the TPL cDNA copies number in the genomic DNA of C1, C2, C3, C4, C5 and C6 clones. Correlation coefficient: 0.993; Slope: -4.100; PCR Efficiency: 75.3%

Figure 4

Purification steps of rTPL. (A) Chromatography of rTPL on Sephacryl S200; the column (2.5 × 150 cm) was equilibrated with 25 mM Tris buffer, pH 8.2, containing 25 mM NaCl and 2 mM Benzamidine (buffer B). The elution of proteins was performed with the same buffer at a rate of 30 ml/h. (B) Chromatography of rTPL on FPLC Mono-Q Sepharose. The column was equilibrated with buffer B; a linear gradient was applied from 25 to 350 mM NaCl in buffer B; the flow rate used was 2 ml/min. (C) Chromatography of rTPL on Sephadex G100; the column was equilibrated in buffer B and the flow rate was 25 ml/h. The pooled fractions containing the rTPL activity were indicated by a dashed line. (D) SDS-PAGE analysis of rTPL performed on 12% gel. Lane1: Low molecular weight marker; Lane 2: culture supernatant; Lane 3: active fraction from Sephacryl-S200; Lane 4: active fraction from Q-Sepharose FF; Lane 5: active fraction from Sephadex-G100.

Figure 5

Recombinant TPL N-terminal sequence.(A) Alignment of native and recombinant TPL N-terminal sequences. (B) Amino acid sequences around the junction of signal peptide (α-factor) and mature TPL.

Figure 6

Effects of temperature and pH on rTPL activity and stability. (A) Effect of temperature on rTPL and nTPL activities. (B) Effect of pH on rTPL and native TPL activities. (C) Effect of temperature on rTPL and nTPL stability. The enzymes are incubated at different temperatures for 30 min. (D) Effect of pH on rTPL and nTPL stability. The enzymes are incubated at different pH for 30 min. Lipase activity was performed under standard conditions.

Figure 7

Effect of increasing concentrations of bile salt (NaDC) and calcium ions (Ca2+) on the rate of hydrolysis of olive oil emulsion by the rTPL. (A) Effect of increasing concentration of bile salt (NaDC) on the rate of hydrolysis of olive oil emulsion by the rTPL and the nTPL. Lipolytic activity was measured at pH 8.5 and 37°C in the absence or in the presence of a molar excess of colipase. (B) Effect of increasing concentration of calcium ions (Ca²⁺) on the rate of hydrolysis of olive oil emulsion by the rTPL and the nTPL. Lipolytic activity was measured at pH 8.5 and 37°C. The star indicates the lipase activity measured in the absence of CaCl2 and in the presence of 10 mM EDTA.