Yeast Expression Vectors

Achieve high levels of expression for your protein, with Gene Synthesis orders and from the ATUM Catalog.

 

Yeast Vectors Elements



Function Pichia S. cerevisiae
Expression Inducible   or   Constitutive Inducible   or   Constitutive
Propagation Targeted genomic integration Yeast self-replicating (2µ) or
Targeted integration
Localization Cytoplasmic   or   Secreted Cytoplasmic   or   Secreted
Integration Protocol Easy, requires linearization Easy
Strains Yes No
IP IP-Free IP-Free

Vector Selector

The methylotrophic yeast Pichia pastoris is a useful system for the expression of milligram-to-gram quantities of protein for both basic laboratory research and industrial manufacturing. ATUM offers IP-Free Pichia vectors with a choice of promoters for cytoplasmic or secreted expression.


Effectiveness of Secretion Signals is Protein Dependent

Efficient protein secretion is highly dependent on the combination of the protein and secretion signal used. We therefore offer vectors with eleven different secretion signals for targeting proteins to the secretory pathway. You can select vectors from the interactive graph below; we recommend testing the entire vector panel to identify the signal that works best with your protein.


 

Yeast Secretion Vector Discount
BuyGet
2 10% off
3 20% off
4 or more 30% off

 

Different secretion signals work well with different proteins. Secreted expression of cutinase and endopeptidase driven by a methanol inducible AOX1 promoter and various secretion signals (pD912-XX). Expression values shown on x- and y-axis are measurements of expressed protein band densities from a SDS-PAGE gel using BSA as standard. In this example, the highest cutinase expression is observed with either alpha-factor secretion signal (AKS) or alpha-Amylase (AA) while the highest endopeptidase expression is observed with Lysozyme(LZ) secretion signal. Expression levels range from 20 to 100 µg/ml for Cutinase and 80 to 450 µg/ml for Endopeptidase; grown in small batch cultures.   More »

Available Secretion Signals




Secretion Signal Amino Acid Sequence Length Promoter Vector
*ATUM vectors with alpha-factor secretion signals FAKS, AKS and AK have protease cleavage sites to help cleave off the alpha-factor protein sequence. The signal peptide (shown in red) is naturally cleaved during translocation and secretion of the protein of interest. The mature alpha-factor protein that remains attached to the protein post-secretion is cleaved by intracellular Kex or Ste proteases.
FAKS – Alpha-factor_full (S.cerevisiae) *MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDL
EGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA
89 aa AOX1, GAP1 pD912, pD915
AKS – Alpha-factor_kex_ste (S.cerevisiae) *MRFPSIFTAVLFAASSALAAPVNTTTEDEL
EGDFDVAVLPFSASIAAKEEGVSLEKREAEA
42 aa AOX1 pD912-AKS
AK – Alpha-factor_kex (S. cerevisiae) *MRFPSIFTAVLFAASSALAAPVNTTTEDEL
EGDFDVAVLPFSASIAAKEEGVSLEKR
38 aa AOX1 pD912-AK
AT – Alpha-factor_T (S. cerevisiae) *MRFPSIFTAVLFAASSALA 19 aa AOX1 pD912-AT
AA – Alpha-amylase (Aspergillus niger) MVAWWSLFLYGLQVAAPALA 20 aa AOX1 pD912-AA
GA – Glucoamylase (Aspergillus awamori) MSFRSLLALSGLVCSGLA 18 aa AOX1 pD912-GA
IN – Inulinase (Kluyveromyces maxianus) MKLAYSLLLPLAGVSA 16 aa AOX1 pD912-IN
IV – Invertase (S.cerevisiae) MLLQAFLFLLAGFAAKISA 19 aa AOX1 pD912-IV
KP – Killer protein (S.cerevisiae) MTKPTQVLVRSVSILFFITLLHLVVA 26 aa AOX1 pD912-KP
LZ – Lysozyme (Gallus gallus) MLGKNDPMCLVLVLLGLTALLGICQG 26 aa AOX1 pD912-LZ
SA – Serum albumin (Homo sapiens) MKWVTFISLLFLFSSAYS 18 aa AOX1 pD912-SA

The major advantage of expressing heterologous proteins as secreted proteins is that Pichia secretes very low levels of native proteins, which means that the secreted heterologous proteins comprise the vast majority of the total protein, facilitating downstream protein purification. Secretion requires the presence of a secretion signal sequence to target the expressed protein to the secretory pathway. While several different secretion signal sequences have been used including the native secretion signals present on some heterologous proteins, success is dependent on the recombinant protein being expressed.


Cytoplasmic Expression with AOX1 and GAP1 Promoters in Pichia

ATUM offers Pichia vectors for cytoplasmic expression with a Zeocin resistance marker and a choice of promoters: methanol inducible AOX1 or constitutive GAP1.

Pichia Vectors Cytoplasmic Expression

DonnerMagenta cytoplasmic expression with GAP1 and AOX1 promoters. Cells were transformed with pD905-DonnerMagenta(GAP1_promoter) or pD902-DonnerMagenta (AOX1_promoter) and plated onto YPD + 300µg/ml Zeocin. Incubate at 30°C until colonies form. For pD905-DonnerMagenta cultures, inoculated single colonies into 25ml of BMGY and incubated at 28-30°C. Samples were taken daily for protein production, beginning 24 hours to 96 hours post inoculation of culture. For pD902-DonnerMagenta cultures, incubated for several days to build biomass at 28-30°C. Induced by removing 10ml of culture and replacing with 10ml of BMM2 with methanol. Boost by adding 125µL methanol twice daily, sampling culture once daily post- induction up to 96 hours. 500µL culture was removed, cells pelleted and lysed in Yeast Busters reagent, mixed with 4X protein sample buffer and 5µL lysate/lane loaded on SDS-PAGE gels. Figure shows both cell pellets as well as lysates run on gel. Controls were untransfected host for construct with GAP1 and non-induced cultures for AOX1 promoter construct. More »


Promoter Properties


Promoter Expression Expression Marker Propagation Localization Vector Map Vectors
* Vectors are recombined into chromosome using SwaI or SacI. Recombination within the chromosome is not localized.
AOX1 Methanol-inducible Zeo Integration* Cytoplasmic
Secretion Signals (11)
Expression Vectors icon See all
GAP1 Constitutive Zeo Integration* Cytoplasmic
Secretion Signals (1)
Expression Vectors icon See all

Advantages:

  • Does not require complex media or culture conditions
  • Genetically easy to manipulate
  • Eukaryotic protein synthesis pathway
  • Can be grown to very high cell densities in minimal media
  • Integrated vectors help genetic stability of the recombinant genes
  • High protein expression levels at the intracellular or extracellular levels
  • Ability to perform higher eukaryotic protein modifications, such as glycosylation, disulphide bond formation and proteolytic processing
  • Available as Electra vectors for rapid cloning and testing protein expression in multiple vectors

Vector Cost

Pichia Vector (pD9xx Electra) Cost for Gene Synthesis Projects: Cloning of synthetic genes into the first vector is available free of charge. If the same gene is cloned into multiple vectors, additional fees will apply. Regular gene synthesis fees apply.

Pichia Vector (pJ9xx) Cost for Gene Synthesis Projects: $200 per vector per order. No additional fees for multiple genes into the same vector in the same order. If the same gene is cloned into multiple vectors, additional fees will apply. Prices are in addition to regular gene synthesis fees.

Positive control constructs are available from the ATUM catalog at half price if ordered with gene synthesis orders.



Protocol

Pichia Culture and Induction Protocol from ATUM

Notes

  • pD and pM vectors (without an ORF) are provided as linearized DNA in solution (10 reactions). pD or pM vectors with a control ORF are provided as circular plasmids (lyophilized).
  • Competent Pichia yeast cell preparation protocol can be found here.
  • Synthetic gene sequence must NOT include SacI (AOX1 promoter) or AvrII (GAP1 promoter) as these sites are used during cloning and linearization. If your gene includes these sites it will be cut internally and not cloned correctly.
  • Vectors with constitutive promoter result in lower expression levels, but do not require addition of methanol.
  • The pJ9xx Pichia vectors were constructed by Anton Glieder, et al at the Graz University of Technology in Austria, for protein expression in P. pastoris.

Vector Selector

ATUM is pleased to offer a selection of IP-Free Pichia pastoris expression strains for research use. These strains are ready to use with all ATUM Pichia expression vectors.

IP-Free© Cell Strains

Strain Properties Datasheet Order
PPS-9010 BG10, wild type
PPS-9011 BG11, aox1Δ (MutS), slow methanol utilization derivative of PPS-9010, useful for fermentation optimization studies
PPS-9016 BG16, pep4Δ, prb1Δ, protease deficient
PPS-KT (Strain Kit) Contains all 3 strains

 

Strain Properties

PPS-9010 (BG10, wild type): Pichia pastoris wild type expression strain. This strain has been characterized by full genomic sequencing and 5000+ gene transcriptome data sets generated for various growth conditions, including shake flask and fermentation cultures. In fermentation, BioGrammatics has achieved cell densities in excess of 700 grams wet cell pellet / liter with this strain.

PPS-9011 (BG11, aox1Δ (MutS)): A slow methanol utilization deriviative of PPS-9010. The AOX1 open reading frame has been deleted from ATG to stop. This strain grows slowly when methanol is used as the sole carbon source, and is most useful for fermentation optimization studies.

PPS-9016 (BG16, pep4Δ, prb1Δ): A protease deletion strain, derived from PPS-9010. The genotype of BG16 is pep4Δ, prb1Δ. Protease deficient strains have been shown to be effective in reducing the degradation of some foreign proteins (White et al. 1995; Brierley 1998). This is especially noticeable in fermenter cultures of secreted recombinant proteins, because the combination of high cell density and lysis of a small percentage of cells results in a relatively high concentration of these vacuolar proteases. Unfortunately, these protease deficient cells are not as vigorous as wild-type strains with respect to PEP4. In addition to lower viability, they possess a slower growth rate, are more difficult to transform, and are more difficult to handle in shake flask and fermenter cultures. Therefore, the use of protease-deficient strains is only recommended in situations where other measures to reduce proteolysis (e.g., addition of carrier compounds such as case amino acids and/or peptone to the culture medium) have yielded unsatisfactory results. In particular, it is recommended that a wild-type P. pastoris strain and not a protease-deficient strain be utilized as the initial strain for expression studies.

These strains have been jointly developed by BioGrammatics Inc. and ATUM.

Proteins and peptides made with ATUM catalog Pichia strains (the “Pichia Strains”) are IP-Free©, and can be used commercially without license obligations. However, neither the Pichia Strains, nor any strains derived from the Pichia Strains, may be transferred or sold to third parties, resold, modified for resale, or used to provide a service of any kind to third parties, including, without limitation, reporting the results of customer activities for a fee or other form of consideration. See the complete legal IP-Free Terms and Conditions here.


Cutinase (in pD912) Expressed in each of 3 Pichia Strains:

 

Pichia cell strains expression

Expression of cutinase (pD912) under control of promoters GAP (upper panel) and AOX1 (lower panel) in three Pichia strains PPS-9010 (wild type), PPS-9011 (aox1Δ (MutS)) and PPS-9016 (pep1Δ, prb1Δ). Non-transformed cells served as negative controls. 50 µl samples were taken daily to follow expression and run on SDS PAGE for cutinase expression. Competent cells were prepared for all three strains and 45 µl cells transformed with 5 µl of linearized DNA. Cultures were grown out for 1 hr on YPDS and plated on YPDS + 1 mg/ml Zeocin and incubated at 30°C. Two or three colonies from each transformed strain were picked and grown in BMGY media (1% yeast extract, 2% peptone, 13.4 g/L YNB, 0.1 M KHPO4 pH 6, 1% glycerol, 0.004 mg/L biotin with 300 µg/ml of Zeocin), supplementing with 1% glycerol each day for 3 days (GAP construct) and with 0.5% (v/v) methanol daily for 2 days (AOX1 construct).   More »


Protocols for Strain Propagation

Strains are in YPD + 1M Sorbitol broth and are shipped at ambient temperature. When received, strains should be stored at 4°C and revived within 1 week by streaking for single colonies to YPD Agar plates (Teknova, Cat. No. Y1000 with 1% yeast extract, 2% peptone, 2% glucose and 2% agar) and incubating for 2 days at 30°C.

Master stocks are made by culturing a single colony of each strain overnight in YPD broth (Teknova, Cat No. Y5000 with 1% yeast extract, 2% peptone, 2% glucose). Harvest cells (5 min at 500 x g) and suspend cell pellet in YPD Broth containing 30% glycerol. Cells are frozen on a dry ice/ethanol bath and stored at -80°C.

Pichia Culture and Induction Protocol from ATUM


Strain Selector

ATUM offers Saccharomyces vectors for cytoplasmic expression with a choice of constitutive (TEF, GPD and ADH) or inducible (GAL1) promoters, and a choice of auxotrophic markers.

Cytoplasmic Protein Expression with Different Promoters

 

 

Cytoplasmic DasherGFP expression with various promoters in S. cerevisiae. DasherGFP was cloned into vectors pD1201 (GAL1_P), pD1211 (TEF_P), pD1221 (ADH_P) and pD1231 (GPD_P), all with Leu2 selectable marker and transformed into S.cerevisiae (MATa/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1). Two clones per construct were grown for 24 hours in complex media, an equivalent amount of culture (approx. 11 OD units at A600) was pelleted. Pellets were resuspended in 250µl Yeast busters reagent. 10µl of lysate was run on SDS-PAGE gel. Quantitation of protein was done by band density compared to BSA as standard. Data is shown as amount of DasherGFP protein expressed per OD unit. Highest constitutive expression was observed with TEF and GPD promoters, ADH is a weak promoter and shows lower expression. Good induction was observed with the GAL1 promoter upon induction with 2% galactose, expression was almost completely repressed in the presence of glucose.   More »

Cytoplasmic Expression of DasherGFP in S. cerevisiae


S. cerevisiae Vectors cytoplasmic expression - DasherGFP

Cytoplasmic Expression of DasherGFP in S. cerevisiae. Genes encoding for fluorescent protein (DasherGFP) were cloned in S. cerevisiae vectors (pJ120x-03_Gal1, pJ121x-03_TEF, pJ122x-03_ADH, pJ123x-03_GPD) and transformed into S. cerevisiae (MATa/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1). Plasmid bearing clones were selected by plating on complete CM glucose agar plates with a single amino acid dropout that corresponds to the amino acid selection marker in the plasmid. Plates were incubated for approximately 3 days at 30°C and photographed under UV light.

 

 

 

 

 

S. cerevisiae Vectors cytoplasmic expression - control

S. cerevisiae cytoplasmic expression control (untransformed cells).


Secreted Protein Expression

Efficient protein secretion is highly dependent on the combination of the protein and secretion signal used, thus ATUM offers vectors with eleven different secretion signals for targeting proteins to the secretory pathway. You can select vectors from the interactive graphs below, either with the strong constitutive TEF promoter, or the weaker ADH promoter. We recommend testing the entire vector panel to identify the signal that works best with your protein.


Effectiveness of Secretion Signal is Protein Dependent


Yeast Secretion Vector Discount
BuyGet
2 10% off
3 20% off
4 or more 30% off
Panel A. Different Secretion Signals Work Well with Different Proteins. Secreted Expression of Cutinase and Endopeptidase driven by a constitutive TEF promoter and various secretion signals (pD1214-XX). Expression values shown on x- and y-axis are measurements of expressed protein band densities from a SDS-PAGE gel using BSA as standard. Highest Cutinase expression is observed with either the alpha-factor secretion signal (AKS) or Invertase (IN). Highest Endopeptidase expression is observed with serum albumin (SA) secretion signal. Expression levels range from 6-85 µg/ml for Cutinase and 4-30 µg/ml for Endopeptidase; grown in small batch cultures.   More »
Panel B. Level of Protein Expression can be Modulated by Promoter Strength. Secreted Expression of Cutinase driven by constitutive TEF and ADH promoters (pD1214-XX and pD1221-XX). Expression values shown on x- and y-axis are measurements of expressed protein band densities from a SDS-PAGE gel using BSA as standard. As observed from the graph, relative expression levels of protein with different secretion signals are not influenced by promoter used. TEF is a stronger promoter and shows higher expression levels, while as a weak promoter ADH may be useful for proteins needed at lower expression levels.   More »

Case Study

Researchers at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) used the ATUM secretion panel to determine the signal with the highest level of expression for their protein.

“In order to select the best signal peptide for secretory expression of GOD, a DNA fragment encoding His-tagged GOD protein was amplified from pDLMOX-GOD(H) (Kim et al., 2006) by PCR using H-GOD-F1 and H-GOD-R1 primers (Supplementary Table S1) and cloned to the Electra vectors of S. cerevisiae secretion signal kit of ATUM (/) according to the protocol provided by the manufacture. After the resulting 11 vectors were introduced to S. cerevisiae L3262 strain, the culture media of the transformants were analyzed by Western blot. Culture medium of the transformant containing pD1214-KP-H-GOD generated the strongest band.”

J Biotechnol 2015. Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification. Gil et al.

Available Secretion Signals




Secretion Signal Amino Acid Sequence Length Promoter Vectors
*ATUM vectors with alpha-Factor secretion signals (FAKS, AKS and AK) have protease cleavage sites to help cleave off the alpha-factor protein sequence. The signal peptide (shown in red) is naturally cleaved during translocation and secretion of the protein of interest. The mature alpha-factor protein that remains attached to the protein post-secretion is cleaved by intracellular Kex or Ste proteases.
FAKS – Alpha-factor_full (S.cerevisiae) *MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDL
EGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA
89 aa TEF, ADH pD12xx-FAKS
AKS – Alpha-factor_kex_ste (S.cerevisiae) *MRFPSIFTAVLFAASSALAAPVNTTTEDEL
EGDFDVAVLPFSASIAAKEEGVSLEKREAEA
42 aa TEF, ADH pD12xx-AKS
AK – Alpha-factor_kex (S. cerevisiae) *MRFPSIFTAVLFAASSALAAPVNTTTEDEL
EGDFDVAVLPFSASIAAKEEGVSLEKR
38 aa TEF, ADH pD12xx-AK
AT – Alpha-factor_T (S. cerevisiae) *MRFPSIFTAVLFAASSALA 19 aa TEF, ADH pD12xx-AT
AA – Alpha-amylase (Aspergillus niger) MVAWWSLFLYGLQVAAPALA 20 aa TEF, ADH pD12xx-AA
GA – Glucoamylase (Aspergillus awamori) MSFRSLLALSGLVCSGLA 18 aa TEF, ADH pD12xx-GA
IN – Inulinase (Kluyveromyces maxianus) MKLAYSLLLPLAGVSA 16 aa TEF, ADH pD12xx-IN
IV – Invertase (S.cerevisiae) MLLQAFLFLLAGFAAKISA 19 aa TEF, ADH pD12xx-IV
KP – Killer protein (S.cerevisiae) MTKPTQVLVRSVSILFFITLLHLVVA 26 aa TEF, ADH pD12xx-KP
LZ – Lysozyme (Gallus gallus) MLGKNDPMCLVLVLLGLTALLGICQG 26 aa TEF, ADH pD12xx-LZ
SA – Serum albumin (Homo sapiens) MKWVTFISLLFLFSSAYS 18 aa TEF, ADH pD12xx-SA

Promoter Properties


Promoter Expression E. coli Markers Expression Markers Propagation Localization Vector Map Vectors
* Vectors are recombined into chromosome using NcoI in the Ura3 marker.
TEF Strong, Constitutive Amp, Chlor, Kan His3, Leu2, Trp1, Ura3, Geneticin 2µ ori Cytoplasmic,
Secretion signals (11)
Expression Vectors icon See all
GPD Strong, Constitutive Amp, Chlor, Kan His3, Leu2, Trp1, Ura3 2µ ori Cytoplasmic Expression Vectors icon See all
ADH Weak, Constitutive Amp, Chlor, Kan His3, Leu2, Trp1, Ura3 2µ ori Cytoplasmic,
Secretion signals (11)
Expression Vectors icon See all
GAL1 Strong, Galactose-inducible,
repressible with 2% glucose
Amp, Chlor, Kan His3, Leu2, Trp1, Ura3 2µ ori,
Integration*
Cytoplasmic Expression Vectors icon See all

Advantages:

  • Does not require complex media or culture conditions
  • Genetically easy to manipulate
  • Eukaryotic protein synthesis pathway
  • Integrated vectors help genetic stability of the recombinant genes
  • Ability to perform higher eukaryotic protein modifications, such as O- or N- linked glycosylation, phosphorylation, disulfide bridge formation, proteolytic processing and folding in a eukaryotic system
  • Available as Electra vectors for rapid cloning and testing protein expression in multiple vectors

One undesirable attribute of S. cerevisiae is the potential to hyperglycosylate proteins, which may hinder the antigenic properties or function of the protein by masking key epitopes or functional sites. Additionally, the hyper-antigenic nature of terminal α, 1, 3 glycan linkages added to expressed protein makes them particularly unsuitable for therapeutic use. Finally, S. cerevisiae generally expresses recombinant proteins at relatively lower levels, as compared to Pichia.



Vector Cost

Saccharomyces Vector Cost for Gene Synthesis Projects: Cloning of synthetic genes into the first vector is available free of charge. If the same gene is cloned into multiple vectors, additional fees will apply. Regular gene synthesis fees apply.
Prices are in addition to regular gene synthesis fees.
Positive control constructs are available from ATUM’s catalog at half price if ordered with gene synthesis orders.



Notes

  • pJ12XX-03C Vectors available as catalog vectors have a DasherGFP® insert, but do NOT contain multiple cloning sites. They can be used as a positive control in conjunction with vectors purchased for gene synthesis orders.
  • For cloning of inserts, use Electra pD12XX catalog vectors.
  • pD and pM vectors (without an ORF) are provided as linearized DNA in solution (10 reactions). pD or pM vectors with a control ORF are provided as circular plasmids (lyophilized).
  • Any vector can be used for gene synthesis.

Vector Selector

Let ATUM Increase Your Expression

Consult with an Expression Specialist today at +1 877 362 8646 or info@atum.bio to achieve your research goals quickly and affordably.