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Location: Home > Custom Services > Protein Related Services > Protein Expression in Yeast

Protein Expression in Yeast

Date: 2018-08-28 Author: Leading Biology Click: 12018

Pichia pastoris expression system introduction

The expression system of Pichia pastoris is a rapidly developed and widely used eukaryotic expression system. It is a methanol nutrition yeast and have two Alcohol oxidase (AOX) coding genes AOX1 and AOX2. The two sequences are similar to each other, but the AOX1 gene is strictly regulated by methanol induction and regulation. When methanol is the only carbon source, the AOX1 promoter can be induced by methanol and initiate the expression of ethanol oxidase, which can be metabolized by methanol. The plasmid containing the AOX1 promoter can be used to promote the expression of the target gene which encoding foreign protein.


Characteristics of expression strain

GS115: Both AOX1 and AOX2 gene could express AOX protein (Alcohol Oxidase), high methanol utilization, close to the wild type, belong to methanol rapid use type (Mut+).

KM71: Most AOX1 gene was knocked out, only AOX2 coding the AOX protein, the rate of using methanol is slow, belong to methanol slow use type (Muts).

different integration of the exogenous gene will cause different phenotypes of expression strains:

Restriction enzyme

 

Insertion site

 

GS115 phenotype

KM71 phenotype

Sa1I/Stul

HIS4

HIS+Mut+

HIS+MutS

Sacl

5’AOX1

HIS+Mut+

HIS+MutS

Bg1II

Replace AOX1

HIS+MutS

HIS+MutS



Common expression vectors and Tags

Expression type

Vector

Tag

Promoter

Intracellular expression

pAO815

HIS4

AOX1

pPIC3K

HIS4

AOX1

pPUC3.5K

HIS4

AOX1

Secretory expression

pPICZα,A,B,C

Bler

AOX1

pPIC9K

HIS4

AOX1


Signal peptide

For extracellular expression proteins, besides the signal peptide carried by the vectors, there are following signal peptides to select:

Signal peptides sequence

Signal peptides

Protein sequence

αMP*

MRFPS IFTAV LFAAS SALAA PVNTT TEDET AQIPA EAVIG YSDLE GDFDV AVLPF SNSTN NGLLF INTTI ASIAA KEEGV SLEKR EAEA

Dse4

MSFSS NVPQL FLLLV LLTNI VSG

Exgl

MNLYL ITLLF ASLCS A

Scw11

MLSTI LNIFI LLLFI QASLQ

Salb

MKWVT FISLL FLFSS AYS


The advantages of Pichia pastoris expression system

1. Contain aspecific powerful AOX(Alcohol oxidase gene) Promoter, the expression of the exogenous gene could be strictly regulated by methanol.

2. Low cost of culture, and easily separated products. The cost of the Pichia pastoris fermentation medium is reasonable, the general carbon source is glycerol, glucose or methanol, the rest is an inorganic salt, there is no protein in the medium, beneficial to the separation and purification of the downstream products.

3. The genetic stability of the extraneous protein gene. Exogenous genes can be integrated into the genome of Pichia pastoris with high copies, the gene is hard to lose and be able to get high expression strains.

4. As a eukaryotic expression system, Pichia pastoris has all the subcellular structures of eukaryotes, could do post-translational modifications, such as glycosylation, fatty acylation, protein phosphorylation, etc.


Advantages of yeast expression system service in Leading Biology

1. Having a complete Pichia pastoris expression platform could design different experimental schemes according to customers requirements.

2. Having a high throughput screening platform could find the best expression conditions quickly and effectively;

3. Could do large-scale fermentation production.


Service Package

Service Items

Time(weeks)

Protein analysis and Codon optimization

Before project starts

Gene synthesis

2-4

Subclone

1

Gene expression and purification

2

Fermentation and purification

1-2

Total

6-9

Classic case

Expression vector: pPIC3.5K (intracellular expression), target protein 32.49 kD (theoretical value)

Expression strain: GS115/KM71

Induction condition: Methanol concentration: 0.5%/1%, induction time 24h/48h/72h

Result analysis: From SDS-PAGE result, the expressions of target protein (40KDa after glycosylation) were observed, GS115-5#-24h, 0.5% methanol, GS115-2#-48h, 1% methanol, GS115-3#-48h, 1% methanol, KM71-1#-72h,1% methanol, each group could show significant band, indicate the target protein is successfully expressed.




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Why Leading Biology?

At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible.

Working with us, you will get stability, and it means a reliable partner to help streamline your R&D process.

Working with us, you will get the guaranteed service to accommodate your requirements.


  • Innovative configurations of chromatography columns custom tailored to each protein
  • Vigorous quality control system to ensure the required quality and reproducibility
  • Production capacity of up to tens of grams
  • Flexible scale-up protein production
  • Competitive price with fast turnaround time

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