IP-Free© Cell Strains

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.

Strain Properties Datasheet Order
PPS-9010 BG10 (wild type)
PPS-9011 BG 11, 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 (pD-912) Expressed in each of 3 Pichia Strains:

 

Pichia cell strains expression

Expression of cutinase (pD-912) 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). 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 10% glycerol each day for 3 days (GAP construct) and with 0.5% (v/v) methanol daily for 2 days (AOX1 construct). Non-transformed cells served as negative controls. 50 µl samples were taken daily to follow expression and run on SDS PAGE for cutinase expression.

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

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.