YeastHIGH<sup>TM</sup> Yeast Expression System - Custom Protein Services

YeastHIGH^TM Advanced Protein Expression Package (SC1346)

GenScript’s engineering-enhanced protein expression technologies enable a high success rate of over 90% over a wide variety of recombinant proteins, and our proprietary YeastHIGH^TM technology is pushing yields even higher. Many secretory proteins that bacterial expression systems yield as insoluble or as inactive inclusion bodies can be produced successfully by our yeast expression system using native or yeast secretion signals.

Milestones	Specifications	Deliverables	Timeline*
1. Subcloning	Subcloning into two appropriate expression vectors Plasmid maxi preparation Linearize the constructs	Construct(s) report	2 weeks
2. Expression optimization and evaluation	Transformation the linear construct into the appropriate host strain Confirmation of positive transformants via PCR Expression optimization and confirmation i: Screening of transformants with multiple gene-insert with in-house YeastFast technology ii: Media formulation and inducer concentrations iii: SDS-PAGE gel and/or Western blot	Expression data	3–5 weeks
3. Protein expression and purification	Small-scale expression from one liter One-step purification, or additional purification to reach purity of 90% or higher	Purified soluble protein QC data	2 weeks
4. Large-scale protein production and purification (Optional)	Large-scale production up to 500 L (Employs fermentors and TFF large-scale filtration system) Deliverables available at >90% purity or higher Removal of tags	Yeast paste/ Purified proteins QC data	2–3 weeks

*Listed is the typical turnaround time at GenScript. Please note that additional requirements may incur additional steps, and likewise longer processing time.

Standard Protein Expression Package (SC1257)

GenScript also provides a standard yeast expression package service, which can be conducted in accordance with any protocol the customer may see fit to supply.

Milestones	Specifications	Deliverables	Timeline*
1. Subcloning	Subcloning into appropriate expression vector Plasmid maxi preparation Linearize the construct	Construct(s) report	2 weeks
2. Expression and evaluation	Transformation of the linear construct into the appropriate host strain Confirmation of positive transformants via PCR Expression confirmation SDS-PAGE gel and/or Western blot	Expression data	2 weeks
3. Pilot expression and purification	1 L yeast culture expression One-step purification, or additional purification to reach purity of 90% or higher	Purified proteins QC data	2 weeks
4. Large-scale protein production and purification (Optional)	Large-scale production up to 500 L (Employs fermentors and TFF large-scale filtration system) Deliverables available at >90% purity or higher Removal of tags	Yeast paste/ Purified proteins QC data	2–3 weeks

*Listed is the typical turnaround time at GenScript. Please note that additional requirements may incur additional steps, and likewise longer processing time.

1. Protein expression in yeast

GenScript's proprietary YeastHIGH^TM expression system and in-house YeastFast technology delivers unparalleled advantages in secretory expression. Pictured below is an enzyme expression performed using our YeastHIGH^TM expression technology. After one step purification by SP-Sepharose, we find tag-free proteins at an expression level of about 20 mg/L and at purity level of over 90%. The target protein is further identified by MALDI-TOF.

Fig. A: Expression and secretion of target protein from P.pastoris
Lane 1 : Conventinal yeast expression technology
Lane 2 : GenScript YeastHIGH^TM technology
Lane 3 : Negative control
Fig. B: Purification of the target protein by SP-Sepharose
Lane M: Smart Protein Standard (GenScript, Cat. No. MM0900)

2. Signal peptides

Shown below is the expression of a protease with signal peptide. There was no secretion expression of target protein with Saccharomyces cerevisiae α factor prepro peptide. After changing the peptide sequence for secretion from α factor prepro to the native signal peptide of the protease itself, the target protein was expressed and secreted successfully.

Fig.       A: Western blot analysis of target protein expression with native signal peptide
Lane 1-5: Induced for 24 h, 48 h, 72 h, 84 h, 120 h
Fig.       B: Western blot analysis of target protein expression with α factor prepro peptide
Lane 1-4: Induced for 24 h, 48 h, 72 h, 84h
Lane    M: Protein Marker

1.YeastHIGH^TM Technology
GenScript YeastHIGH^TM technology harnesses the strongest advantages of yeast expression, including promoters, optimal copies of vector per cell, inducibility, signal peptides, and cellular location, allowing us to provide customers with high-level recombinant protein production. During our years in the yeast protein expression service field, we have successfully delivered hundreds of proteins, including enzymes, proteases, kinases, cytokines, receptors, and transcription factors, and among others.

2. OptimumGene^TM codon optimization technology for gene expression
GenScript's advanced OptimumGene^TM codon optimization technology can optimize your gene sequence, allowing it to reach the highest possible level of gene expression and protein solubility. The OptimumGene^TM algorithm optimizes a variety of parameters that are critical to transcription, translation, and protein folding. Learn more...

3. Large-scale protein production
GenScript's fermentor facility provides high-quality protein production, for orders of up to 500 L. Our TFF large-scale filtration system can enhance purity and yield.

4. Purification
GenScript offers a variety of flexible purification systems: affinity column, gel filtration, ion exchange, hydrophobic-interaction column, and HPLC. Various tags are added to protein to facilitate their purification and we also offer services for tag removal.

5. Competitive prices
Multiple yeast expression packages are supplied at competitive prices, starting from $2,900, to fit customer’s budget.

Yeast Expression System

Quotations and Ordering: