YLEX Yeast Expression Kit

Product overview

Description
YLEX Expression Kit based on INRA INAPG licensed patent* provides an easy approach for cloning and expressing a gene of interest in the yeast, Yarrowia lipolytica. Using this kit, high level of heterologous protein may be expressed intracellularly or be secreted from the cell into the medium by selecting the supplied expression vector pYLEX1 or pYLSC1.

 

* INRA (Institut National de la Recherche Agronomique) and INAPG (Institut National Agronomique Paris-Grignon)

Features
    • Safe: Y. lipolytica was classified as GRAS (Generally Regarded As Safe) by the US FDA (Food and Drug Administration)
    • Simple: a simple tool for expressing heterologous protein
    • Easy manipulation: like E. coli and S. cerevisiae
    • Stable: strong stability in vectors and constructed plasmids
    • Reliable: vectors integrated at the same site in genome
    • Flexible: both expression and secretion vector provided (proteins may be expressed intracellularly or be secreted from the cell into medium)
    • High growth ability: high secretion capacity & high product yield
    • Less protein degraded: no extracellular protease synthesized by a special protease-deficient Yarrowia strain
    • Mass production: industrial mass production of recombinant proteins
    • Less hyperglycosylation:able to perform post-translational processing of complex proteins, unlike S. cerevisiae
Applications
Heterologous protein expression, either intracellular or extracellular depends on selected vector in GRAS yeast.
Yarrowia Vectors
Two vectors (pYLEX1 and pYLSC1) are included in this kit, and they can be used for either intracellular expression or secretion of proteins of interest in Y. lipolytica. Generally speaking, if the target protein is cytosolic and non-glycosylated, the pYLEX1 vector is a better choice. If the protein of your interest is normally glycosylated or secreted, you may want to choose the pYLSC1 vector.
The pYLEX1 expression vector (7259 bp) contains the strong hybrid promoter (hp4d) carrying four tandem copies of upstream activator sequences (UAS1B) fragment from pXPR2 and a minimal pLEU2 fragment. The multiple cloning sites and the XPR2 transcription terminator lie immediately downstream of 3′ site of hp4d promoter, followed by a leucine selection marker gene (LEU2). The vector can be linearized by digestion with NotI to create a linear DNA fragment capable of inserting into the Y. lipolytica genome.
The pYLSC1 secretion vector (7205 bp) contains the hybrid promoter (hp4d) and a secretion signal (XPR2 pre region). The multiple cloning site and the pXPR2 transcription terminator lie immediately downstream of 3′ site of XPR2 pre region, followed by a leucine selection marker gene (LEU2). The vector can be linearized by digestion with NotI to create a linear DNA fragment capable of inserting into the Y. lipolytica genome.
Yeast Strain
The strain Po1g of Yarrowia lipolytica is a derivative of the wild-type strain W29 (ATCC 20460) by a series of genetic modifications. Briefly, the original URA3 gene in the W29 strain was disrupted with the SUC2 gene from Saccharomyces cerevisiae, followed by the introduction of a deletion in the LEU2 gene. Furthermore, the deletion of the XPR2 and AXP genes ensures that Po1g is unable to produce any extracellular protease. In order to allow easy integration of pBR-based expression/secretion vectors, a pBR322 docking platform was integrated at the URA3 locus.
Related Product

YLOS Yeast Transformation Kit

Specifications

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Stain & Genotype
Strain Genotype Phenotype
Po 1 g MatA, leu2-270, ura3-302::URA3,
xpr2-332, axp-2		 Leu-, ΔAEP, ΔAXP, Suc+, pBR platform

Specifications
# FYY201-1KT
Yarrowia lipolytica Yeast Strain: Po1g 1 stab
pYLEX1 – Expression Vector 5 μg
pYLSC1 – Secretion Vector 5 μg
Primer 6560 F 250 μl
Primer 6904 R 250 μl
YLOS Transformation Kit
(# FYY301-120P)		 1 kit
* YLEX Expression Kit is produced by Yeastern Biotech Co., Ltd. with patent protected, which is authorized with the non-exclusive manufacturing and distribution worldwide by the proprietary property of Institute National de la Recherche Agronomique (INRA), France.

Technical

Plasmids

Vectors provided in the YLEX contain a strong hybrid promoter carrying four tandem copies of upstream activator sequences (UAS1B) fragment from pXPR2 and a minimal pLEU2 fragment. Unlike the frequently used Yarrowia promoter (pXPR2), this stable hybrid promoter directs protein expression constantly without multiple influences by nutritional and environmental factors in the medium.

When a constructed plasmid with the hybrid promoter followed by a cloned gene of interest is linearized by the selected restriction enzyme, it becomes an expression cassette that can integrate into the Y. lipolytica genome by homologous recombination within the process of transformation. The successful transformants are ready for expression or secretion of recombinant protein depending on whether secretion signal appears on the plasmid. For more information, please read the articles cited in this catalog.

Protocol
Quality Control
Two plasmids, pYLEX1+AMY1 (mouse salivary a-amylase gene) and pYLSC1+AMY1Δ (AMY1 without its native secretion signal) are used to ensure functional transformation kit and correct gene expression in the host. Contamination test is also performed to ensure no other microbial contamination.
Experimental Data
The figure shows that filtered culture medium from a batch culture of both amylase-encoding transformants (YL #2 and #3) could digest starch in solid medium agar, and subsequently produce clear zones. In contrast, medium from the culture of yeast transformed with vector only (YL #1) did not exhibit the same result. It indicates that cloning of both a-amylase gene (AMY1) into pYLEX1 and a-amylase gene without its secretion signal peptide (AMY1D) into pYLSC1 was successful by using the YLEX Expression Kit. In both cases, active a-amylase was efficiently secreted into the culture medium.
A time course study of the capacity of high cell density fermentation and secretion of recombinant enzyme in the YLEX system (Yarrowia lipolytica expression system).
Overproduction of heterologous protein (SDS-PAGE analysis): Secretion profile of recombinant enzyme in the supernatant, samples were collected by time course, 10 μL/lane.
High cell density fermentation: High cell density fermentation in a 14L fed-batch fermenter, cells were centrifuged (3,000 rpm) by time-course.

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