Yeast Expression System

Introduction:

Yeast protein expression system is a highly economical eukaryotic expression system that do both secretion expression and intracellular expression. The exogenous gene expressed by Yeast expression system has a certain post-translational processing capacity, the expressed exogenous protein has a certain degree of folding and glycosylation modification, it’s more stable than prokaryotic expressed proteins, particularly suitable for the expression of eukaryotic genes and preparation of functional proteins. Yeast secretion expression can secrete the expressed exogenous protein into the extracellular matrix, so that it is easy to obtain a high purity protein. Yeast expression system has many advantages which make its research and application more and more widely.

Advantages:
  • Cost-effective, high expression level: as high as 100 mg/L (Shake flask culture)
  • No self-produced endotoxin
  • Products have post-translational modifications: glycosylation, phosphorylation, acylation, etc., it is more likely to have biological activity
  • Products can be properly folded and efficient secretion
  • It is more stable than prokaryotic proteins, and it is particularly suitable for the expression and preparation of functional proteins such as tuberculosis proteins, defensin, interleukin and cytokines
  • With patented Biobrick technology, we can achieve efficient in vitro construction of any copy of the gene dose
  • Unique PickRight technology, thus can directly obtain high expression level strains without screening after transformation, the time compared to traditional screening reduced 5-10 business days
  • Price as low as $780, delivery time as short as 35 business days
Guarantee:

Risk-free: We do NOT charge if we cannot deliver the protein.

Service Process:

Steps Project Process Cusabio Features Lead Time
1 Expression vector construction Codon optimization; gene synthesis Multiple vectors optimization, More options for customers
In order to improve the success rate of expression and achieve higher yield, in addition to conventional N-terminal fusion protein expression, we also provide protein with C-terminal fusion label, in greater degree to ensure the activity while ensuring the purity.
15-20 business days
The PCR amplification products are ligated to the expression vectors e.g. pPic9k, pPic3.5k, pPiczαA, etc.
Transform ligation mixtures into E. coli strain
Obtain the correct recombinant plasmid
2 Transformation and strain identification Prepare the recombinant plasmid in large quantities High copy screening
Conduct multiple screening through unique screening markers of different vectors, and the highest expression level strain was gradually obtained.
PickRight Technology
The high expression level strain was obtained directly after transformation, and the time was shorten by 5-10 business days compared with the traditional screening. (Theoretically this technology is mainly recommended for the production of less than 5 mg/L protein expression)
10-13 business days
Linearization of recombinant plasmid
Transform to GS115, X33, KM71 and other hosts by electroporation
PCR analysis is recommended to verify successful transformants
Use geneticin G418, Zeocion and other antibiotics for multiple copies screening to obtain high copy
3 Small test, scale up expression and purification Small scale expression screening (20-40 strains) 7-12 business days/15-25 business days (Featured Purification)
Determine strain and optimize expression conditions
Scale up culture
Protein purification Multiple purification methods (optional)
Explore different chromatographic conditions including ion exchange, hydrophobic and others by using AKTA, and then determine the optimal purification method.
4 Additional services (Optional) Charge Tag removal service Flexible additional services
Customers can flexibly choose from a variety of additional services to their specific needs, e.g. Endotoxin removal, Filter-sterilization, Tag removal, Lyophilization, etc. Some are complimentary, and some require additional charge.
3 business days
Free Filter-sterilization; Endotoxin removal; Lyophilization
(Note: Lyophilization and filter-sterilization can not be met simultaneously)
2 business days
5 Quality Control Testing of purity, concentration, etc. QC report is provided. Detailed COA Report
Detailed product data sheet and COA are provided for each project.
3-5 business days
Total lead time 35-50 business days

Case 1 High Purity Protein
Difficulty: Yeast intracellular expression, many impurities can be observed in the lysate protein (lane 1), the target protein was not obvious, after the yeast system-specific chromatography system purification, less miscellaneous band was observed with SDS-PAGE detection, the purity reached more than 95% (lane 6-7).

  • Lane 1:Lysate
  • Lane 2:Marker
  • Lane 3:Flow through
  • Lane 4-7:Target protein by different gradient elution
  • Lane 1:Lysate
  • Lane 2:Flow through
  • Lane 3:Marker
  • Lane 4-7:Target protein by different gradient elution

Features: Yeast secretion expression, the expressed protein was directly secreted into the medium, basically no impurities, the purity is as high as 90% or more, the purification is mainly for removing pigment and other residues in the medium, leaving only the target protein in the appropriate buffer.

  • Lane 1:Culture medium supernatant
  • Lane 2:Marker
  • Lane 3:Flow through
  • Lane 4:The eluted target protein

Case 2: Large Molecule Weight Protein Expression
Difficulty: More than 700 amino acids, Mw: 81 kDa, we chose secretory vector for expression in order to get a higher purity, finally the protein was successful expressed and secreted into the culture medium.

  • Lane 1:Culture medium supernatant
  • Lane 2:Flow through
  • Lane 3:The eluted target protein
  • Lane 4:Marker

Case 3: Small Molecule Weight Protein Expression
Difficulty: 62 amino acids, Mw: 7 kDa, the molecular weight is very small, relatively difficult to concentrate and detect the protein. The expression, purification, collection were very successful from the SDS-PAGE detection result.

  • Lane 1:Culture medium supernatant
  • Lane 2:The eluted target protein
  • Lane 3:Marker

Difficulty: www 36 amino acids, Mw: 4 kDa, and this customer required to remove the tag, it’s rather difficult to remove the tag as the molecular weight itself is very small, but we have successfully remove the tag after a series processing, and WB detection result showed the target protein didn’t contain tag, so finally high-purity, low-molecular-weight and untagged protein was obtained.

Case 4: N-linked glycosylation modification (Yeast expression system unique characteristics)
Features: Close to the modification of native proteins, especially glycosylation in the Yeast expression system is particularly evident by SDS-PAGE detection, it showed diffuse band and a large molecular weight, after digested by Endo H, the band was shaped and the size is consistent with the theoretical value.

  • Lane 1:Purified target protein (N-linked glycosylated modification)
  • Lane 2:Protein digested by Endo H enzyme
  • Lane 3:Marker

Case 5: pPic9k-SUMO Vector
Unique SUMO tag fusion protein

  • Lane 1:Culture medium supernatant
  • Lane 2:Flow through
  • Lane 3:Marker
  • Lane 4:SUMO tag fusion protein

Characteristic expression systems

pPic9k-JT Plasmid+GS115 strain efficient expression

  • 1.

    Carrys AOX1 strong promoter, containing alpha secreting factor which is able to do high efficient secretion expression

  • 2.

    Seamless cloning, no restriction enzyme needed

  • 3.

    Multiple linearization sites:SalⅠ, SacⅠ, BglⅡ

  • 4.

    Can do Amp and Kan double-resistant screening to select positive strains during the prokaryotic stage

  • 5.

    Can do His+ and G418 screening to select high expression level strains during the eukaryotic stage

  • 6.

    Modified cloning sites, its cloning is not limited by the potential endonuclease in the target gene

  • 7.

    The vector has his tag, thus making it easy for cloning and purification

pPic9k-SUMO Plasmid+GS115 strain efficient expression

  • 1.

    Carry AOX1 strong promoter, containing alpha secreting factor which is able to do high efficient secretion expression

  • 2.

    Contain SUMO fusion protein; possessing a strong ability to promote expression

  • 3.

    Seamless cloning, no restriction enzyme needed.

  • 4.

    Contain the EK cleavage site, can obtain untagged protein after enzyme digestion.

  • 5.

    Multiple linearization sites:SalⅠ, SacⅠ

  • 6.

    Can do Amp and Kan double-resistant screening to select positive strains during the prokaryotic stage

  • 7.

    Can do His+ and G418 screening to select high expression level strains during the eukaryotic stage

  • 8.

    Modified cloning sites, its cloning is not limited by the potential endonuclease in the target gene

  • 9.

    The vector has his tag, thus making it easy for cloning and purification

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