pGAPZA-α-MF-op-rLlAlp2

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	Clones transformed with construct V and VI were continuously grown in indicated media (50 or 25 mL) for 2 d. Aliquots of 1 mL of growth medium containing secreted active enzyme were taken daily, centrifuged at 10,000 × g for 2 min at 4 °C and dialyzed against Tris–HCl buffer (10 mM, pH 7.4) for about 20 h and enzymatic activities determined.

Protocol tips
Clones transformed with construct V and VI were continuously grown in indicated media (50 or 25 mL) for 2 d. Aliquots of 1 mL of growth medium containing secreted active enzyme were taken daily, centrifuged at 10,000 × g for 2 min at 4 °C and dialyzed against Tris–HCl buffer (10 mM, pH 7.4) for about 20 h and enzymatic activities determined.

Publication protocol

"The expression of LlALP2 in P. pastoris and the extraction of the extracellular and intracellular enzyme were performed as follows: a single colony selected on YPD agar plates containing Zeocin (0.10 mg/mL) was inoculated in BMGY medium (5 mL) and grown at 30 °C overnight. Aliquots of 2 mL starter culture were added to various culture media (50 mL in 125 mL baffled flask covered with sterile cheese cloth, if secreted expression 1.00% casamino acid included) as indicated. In case of clones transformed with construct I, II, III and IV, cells were collected after 1 d by centrifugation at 3000 × g for 5 min and resuspended in BMMY (20 mL, with 1% casamino acid) and grown for 2 d at 20.5 ± 0.5 °C. Sterile distilled deionized H2O (1.8 mL) and methanol (0.2 mL) were supplemented every 24 h (10% of the culture volume) to BMMY medium. Clones transformed with construct V and VI were continuously grown in indicated media (50 or 25 mL) for 2 d. Aliquots of 1 mL of growth medium containing secreted active enzyme were taken daily, centrifuged at 10,000 × g for 2 min at 4 °C and dialyzed against Tris–HCl buffer (10 mM, pH 7.4) for about 20 h and enzymatic activities determined. Cell pellets and supernatant were collected separately after 2 d growth by centrifugation at 10,000 × g for 5 min and wet cell mass was recorded.

The cell pellets were resuspended in lysis buffer (Tris–HCl, 50 mM, pH 7.4; PMSF, 1 mM; 2 mL per g). Aliquots of 1 mL of resuspended cell pellets were added to 0.5 mm zirconia/silica disruption beads (0.5 mL, Research Products International Corp., Mt. Prospect, IL) and lysed with Vortex Genie 2 for 30 s by vigorous mixing followed by 30 s incubation on ice. This mixing/freezing cycle was repeated for a total of 6 cycles. Portions containing intracellular enzyme were collected by centrifugation at 12,000 × g for 15 min at 4 °C and 1 mL aliquots of supernatant containing secreted active enzyme was dialyzed against Tris–HCl buffer (10 mM, pH 7.4) for 20 h to remove phosphates and salts before assaying for enzymatic activity."

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