Journal: Biotechnology Reports

Article Title: Extracellular expression of alkaline phytase in Pichia pastoris : Influence of signal peptides, promoters and growth medium

doi: 10.1016/j.btre.2015.03.005

Figure Lengend Snippet: Map of rLlALP2 expression constructs. P AOX1 and P GAP , P. pastoris AOX1 and GAP promoter; α-MF, α-MF secretion signal peptide; N, 19 aa signal peptide at N-terminus; op-rLlAlp2 , cDNA of optimized alkaline phytase; C, five amino acid residues (QKTEL); AOXTT, AOX1 transcription termination region; P T , S. cerevisiae transcription elongation factor 1 promoter that drives expression of Sh ble gene in P. pastoris ; P E , promoter that drives expression of Sh ble gene in E. coli ; Zeo r , Zeocin resistant gene from Streptoalloteichus hindustanus bleomycin gene ( Sh ble ) and CYC1TT, CYX1 transcription termination region. I, full length optimized rLlAlp2 sequence ( pPICZαA-op-rLlAlp2 ); II , truncated op - rLlAlp2 without QKTEL at the C-terminus ( pPICZαA-op-LlAlp2ΔC ); III, truncated op - rLlAlp2 without the first 19 aa at the N-terminus and without QKTEL at the C-terminus ( pPICZαA-op-rLlAlp2ΔNΔC ); IV , op-rLlAlp2-ΔNΔC was introduced downstream of CL ( pPICZA-CL-op-rLlAlp2-ΔNΔC) ; V , pGAPZA-op-rLlAlp2 driven by P GAP ; and VI , extracellular expression directed by α-MF ( pGAPZA-α-MF-op-rLlAlp2 ).

Article Snippet: Escherichia coli TOP10F’, P . pastoris strains X-33 and expression vector pPICZA were purchased from Invitrogen (Carlsbad, CA).

Techniques: Expressing, Construct, Sequencing

Journal: Biotechnology Reports

Article Title: Extracellular expression of alkaline phytase in Pichia pastoris : Influence of signal peptides, promoters and growth medium

doi: 10.1016/j.btre.2015.03.005

Figure Lengend Snippet: Expression level of rLlALP2 driven by P GAP . Panel A: intracellular expression levels and copy numbers of rLlAlp2 in clones transformed with construct V . A total of 10 clones were randomly selected and grown in YPD medium (50 mL) at 29 ± 0.5 °C for 48 h and cell pellets were collected and lysed; Panel B: effect of growth medium on extracellular and intracellular expression levels of rLlALP2 in P. pastoris clones transformed with construct VI . The best producer (with copy number one) was selected and grown on various media (YPD + G: 1.00% glucose and 1.00% glycerol, YPDS: 1.00% glucose and 1 M sorbitol, BMGY 4, 6 and 7.5: 1.00% glycerol, pH 4.0, pH 6.0 and pH 7.5; BMGY Triton: 1.00% glycerol, 0.01% Triton X-100 and BMGHY: 1.00% glycerol, 0.01% Triton X-100 and 0.004% histidine) as indicated at 20.5 ± 0.5 °C for 48 h; Panel C: effect of promoters and copy number of rLlAlp2 on extracellular expression level of rLlALP2 in P. pastoris clones, transformed with expression vector pGAPZαA-op-rLlAlp2-ΔNΔC (construct VI ) and pPICZA-α-MF-op-rLlAlp2-ΔNΔC (construct III ) . Seven clones transformed with construct VI were randomly selected and grown in BMGHY Triton (50 mL) at 20.5 ± 0.5 °C for 48 h. Extracellular medium from time = 0 cultures were used as controls. Six clones transformed with construct III were grown as described in . Extracellular medium from non-induced cultures were used as controls.

Article Snippet: Escherichia coli TOP10F’, P . pastoris strains X-33 and expression vector pPICZA were purchased from Invitrogen (Carlsbad, CA).

Techniques: Expressing, Clone Assay, Transformation Assay, Construct, Plasmid Preparation