Heterologous expression of alkaline phytase from lily pollen in bacteria and yeast

Phytases are enzymes that catalyse the hydrolysis of myo-inositol hexakisphosphate (phytic acid). Alkaline phytase is a homodimeric protein (116 kDa) with a total of 20 Cys residues. This dissertation describes a multipronged approach to achieve the heterologous production of soluble, active alkaline phytase.;In attempts to produce soluble, active enzyme, recombinant alkaline phytase (rLlALP2) was expressed in a variety of E. coli strains that differ in cellular characteristics such as proteases (HB101), IPTG uptake (Tuner(TM)), and tRNA availability (Rosetta(TM)). In all strains investigated no activity was detectable in the soluble form; all strains showed alkaline phytase activity in insoluble lysate. rLlALP2 was then expressed in the E. coli strain Origami(TM), which contains mutations in the thioredoxin reductase and glutathione oxidoreductase genes. This renders the cytoplasmic environment less reducing and therefore allows for increased disulfide formation and proper folding. Growth of Origami(TM) at lower temperatures (18°C) to increase partitioning of rLlALP2 in the soluble form did lead to an increase in activity in the soluble lysate (17% versus 0.7% at 37°C); a majority (83%) was still found in the insoluble lysate. Therefore, efforts to solubilize and refold the enzyme in inclusion bodies were examined. Solubilization with urea (2 M) and glutathione (0.5 mM) and refolding in Tris-HCl (10 mM, pH 7.0) and glutathione (0.5 mM) was able to refold a small portion of rLlALP2 (12%); most of the enzyme could not be converted to the soluble active form.;A second strategy to produce the enzyme in the soluble form was to express alkaline phytase as a fusion protein with a soluble protein partner, either maltose binding protein or glutathione S-transferase. SDS-PAGE and Western blotting showed the production of the soluble, fusion proteins in E. coli. However, the fusion protein was not active, possibly due to improper folding.;Another approach was to express rLlALP2 in the eukaryotic yeast Pichia pastoris. Recombinant expression in eukaryotes can often lead to soluble, active enzymes due to folding and post-translational modifications in the endoplasmic reticulum and golgi. Prokaryotes lack these organelles and therefore misfolded heterologous proteins often accumulate in inclusion bodies. Expression in P. pastoris yielded soluble, active alkaline phytase. Catalytic characterization showed that rLlALP2 had properties similar to native alkaline phytase suggesting that the enzyme was properly folded. However, total activity levels were low; expression in clones ranged from undetectable levels to 2000 U/25 mL.;In an effort to increase yields of rLlALP2 in P. pastoris, the cDNA coding for alkaline phytase was optimized; codons were changed to suit the Pichia codon bias, creation of stable RNA secondary structures was prevented, and a premature transcriptional terminator were removed. Expression utilizing optimized LlAlp2 cDNA in P. pastoris produced increased yields of rLlAlP2, including several clones (7/23) with high alkaline phytase activity (> 1000 U/25 mL). Clones were screened on varying concentrations of the antibiotic zeocin because increased zeocin resistance has corresponded to increased expression of recombinant proteins. Colonies selected on lower levels of zeocin (100 mug/mL) had more clones producing high levels of rLlALP2 activity (7/16) compared to clones selected on higher levels of zeocin (1000 mug/mL, 0/7).;In order to screen large numbers of clones on agar plates rapidly, a colorimetric, in situ LlALP2 assay was developed. Cell lysates from rLlALP2-producing colonies produced a blue precipitate that was proportionate to enzyme activity when added to 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium embedded agar plates. The assay can be employed to screen for colonies secreting rLlALP2.;We describe the heterologous expression of active soluble rLlALP2 in Pichia pastoris. Pichia pastoris can serve as a potential source of rLlALP2 for structural and animal studies. (Abstract shortened by UMI.)...