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View additional product information for EasySelect™ Pichia Expression Kit - FAQs (K174001)
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次のプロトコールがPichia pastorisで良く使用されています。250 mLの培養液を使用しますが、1 mLまでスケールダウン可能です。
1. 10 mL YPD培地にPichiaを植菌し、30℃で一晩振騰培養してください。
2. 翌朝、OD600を測定してください。午後にログフェーズになるように培養液を希釈してください（午後4-5時位にDO600 = 3.0が目安）。
3. OD600が約3.0になったら、250 μL培養液を250 mL YPDに植え継いでください。翌朝元気なログフェーズにあるOD600 = 1.0付近の細胞を準備することが目的です。
4. OD600が約1.0になったら、1 L遠心管で、3,000 x gで10分間遠心分離してください。
5. やさしく250 mLの冷やした蒸留水に細胞を再懸濁してください。
6. 500 mLの遠心管に移し、3,000 x g、10分の条件で再度遠心分離してください。
7. 20 mLの冷やした1 Mソルビトール溶液に再懸濁し、50 mLのチューブに移してください。
8. 3,000 x g、10分間遠心分離してください。
9. 1 mLの1 Mソルビトール溶液に再懸濁し、氷上で保持してください。
10. 上記80 μLを1回のエレクトロポレーションに使用してください。
抗生物質の使用はお薦めできません。少なくともアンピシリン、カナマイシンの添加により発酵過程に問題が生じることはありませんが、発酵時にpHが低下するため抗生物質は不活化されます。最も優れた方法は無菌操作を確実に行う事です。
通常酸によるpH調整の必要はありません。発酵過程で培地は酸性化する傾向があります。一方、培地のpHが上昇した場合は、炭素源の枯渇、健康状態の悪化が予測されます。
Pichia発酵における窒素源はpH調整に使用される水酸化アンモニウムです。発酵に使用される基礎培地、無機塩類には窒素源は含まれません。
弊社製品（catalog # Q30007）をご利用いただく場合、それぞれのNB、S. cerevisiae用の1 L 10X YNBを調製可能です（Pichia培地用はS. cerevisiae用の2倍濃度です）。以下は1X YNBの組成です:
Ammonium Sulfate................. 5 g
Biotin....................... 0.002 mg
Calcium Pantothenate........... 0.4 mg
Folic Acid................... 0.002 mg
Inositol......................... 2 mg
Niacin......................... 0.4 mg
p-Aminobenzoic acid............ 0.2 mg
Pyridoxine HCl................. 0.4 mg
Riboflavin..................... 0.2 mg
Thiamine HCl................... 0.4 mg
Boric Acid..................... 0.5 mg
Copper Sulfate.................0.04 mg
Potassium Iodide............... 0.1 mg
Ferric Chloride................ 0.2 mg
Magnesium Sulfate.............. 0.4 mg
Sodium Molybdate............... 0.2 mg
Zinc Sulfate................... 0.4 mg
Potassium Phosphate Monobasic...1 g
Magnesium Sulfate.............. 0.5 g
Sodium Chloride................ 0.1 g
Calcium Chloride............... 0.1 g
AOX1、AOX2共に72,000（Ellis, et. al. 1985）。
使用するクローンがMut+かMutSかに依存します。
Mut+の場合、最初（最初の2-4時間の誘導期間）培地の酸素取り込み速度はグリセロールバッチフェーズの最終段階よりも遅いと予測されます。その後メタノールに適応した後、良好な状態（例えば、メタノール量が多すぎた場合に起こる毒性が出ていない）であれば、酸素取り込み速度は顕著に増大します。なお、Mut+クローンを用いた発酵を行う場合、メタノールスパイクテストを行うことをお薦めします。
MutSの場合、発酵過程を通して酸素取り込み速度は、グリセロールバッチフェーズの最終段階よりも遅いと予測されます。なお、MutSクローンを扱う場合、よりメタノールの毒性に注意を払う必要があります。
タンパク質が分泌経路（αファクターリーダー配列やその他の分泌シグナルが通過に関与します）を通る場合、糖鎖付加が起こる可能性があります。しかしながら、糖鎖付加が行われるためには、タンパク質の表面にN結合型、O結合型糖鎖付加コンセンサス配列が存在する必要があります。
PTM1塩、あるいは発酵用培地に硫酸を添加する必要はありません。多少塩類の溶解性を高める可能性はありますが、それ以外のメリットはありません。
αファクター分泌シグナルはS. cerevisiae由来で、P. pastoris、K. lactisを含む多くの酵母種で機能します。
GS115/pPICZ/lacZ、GS115/HAS、GS115をヒスチジンを含む最少培地にストリークしました。炭素源はメタノールです。GS115/pPICZ/lacZとGS115は上記メタノールプレート上での生育が早く、一方GS115/HASは非常に遅いことがわかりました。この生育速度の違いから GS115/pPICZ/lacZはMut+、GS115/HASはMutSだと判断しました。なお、フレッシュなヒスチジンの使用がこの実験において非常に重要です。
可能です。必要であれば、選別の際にYPDプレートにZeocin溶液を塗布することは可能です。3 mm径ガラスビーズ10-15個を用いて塗布した場合うまくいったという報告があります。ただし、塗布することにより抗生物質の効果が薄まる可能性があるので、ある程度の最適化は必要だと考えてください。
幾つかご注意いただきたい点があります。
（1）高いODになるまで培養した細胞を使用した場合、うまくスフェロプラストはできません。培養しすぎた細胞の使用は避けてください。
（2）古い細胞は使用せず、ログフェーズにある細胞を使用してください。
（3）zymolaseは使用前によく混ぜてください。Zymolaseは溶媒に完全に溶けているわけではありません（懸濁液です）。
（4）PEG溶液が新しいこと、pHが正しいことをチェックしてください。
可能です。YPDでも問題ありませんが、2点問題があります。1点目は、YPD培地を使用した場合泡が出来やすく、その制御は非常に困難です。2点目は、YPD培地は非常にリッチな培地なので、分泌タンパク質の精製が困難なことがあります。BMGY培地は上記2点においてYPDよりも有利です。
当社のプロトコールで使用されているyticaseは粗精製酵素です。高純度のものは高価ですし、必要ありません。我々は、本プロトコールで細胞溶解のためにzymolaseも使用しています（1 mg/mLストック溶液を5 μL使用）。これは過剰量ですが、非常に有効です。
Ref: BioTechniques 20:980-982 June 1996.
αシグナル配列（αシグナル配列とプロホルモンリーダー配列を含む）はPichia細胞内で3種の酵素によって4度切断されます。まず、N末端側で、シグナルペプチダーぜで切断され、次にマルチクローニングサイトの少し上流のLys-Arg配列の下流でKex2により切断され、最後にGlu-Alaの連続配列でSte11により2度切断されます。
使用できます。Pichiaで発現させたHisタグ融合タンパク質の精製にProBond Purification systemを使用可能です。Pichia培養上清からProBondを用いてタンパク質精製を行う際の注意点を以下に示します。
1. Pichia培養上清のpHを7.5-8.0に調整してください。
2. デカントで上清と白い沈殿を分けてください（希に発現タンパク質が沈殿していることがあるので、念のため沈殿は保管しておくことをお勧めします）。
3. カラムが詰まるのを防ぐために、細胞残渣などの沈殿を遠心分離で除去してください。
4. 伝導率を500 mM NaCl相当になるように塩を添加して調整してください（高塩濃度培地を使用している場合は必要無いかもしれません）。
5. カラム精製操作をマニュアルに従って行ってください。
多くの研究者は消泡剤としてMAZU DF 204あるいはKFO 673を使用します。Antifoam 289も使用できるかもしれませんが、効果はあまり長続きしません。できるだけ少ない量の消泡剤を使用してください。正常な5 L培養液に対して0～数mLの消泡剤の使用が適当です。よく制御された消泡剤コントローラーを用いて添加してください。なお、培養液の泡立ちが激しい場合は、炭素源が枯渇している、pHが低くなっている、培養状態が健全ではないことを示している可能性があります。
必要であれば以下のコドン使用頻度リストを使用してください：
Glycine: GGT or GGA
Glutamic acid: GAG or GAA
Aspartic acid: GAC or GAT
Valine: GTT or GTC
Alanine: GCT or GCC
Arginine: AGA or CGT
Serine: TCT or TCC
Lysine: AAG
Aspargine: AAC
Methionine: ATG
Isoleucine: ATT or ATC
Threonine: ACT or ACC
Tryptophan: TGG
Cysteine: TGT
Tyrosine: TAC
Leucine: TTG or CTG
Phenylalanine: TTC
Glutamine: CAA or CAG
Histidine: CAC or CAT
Proline: CCA or CCT
HASは非還元SDS-PAGEでは55 kDa付近に、還元SDS-PAGEでは66 kDa付近にバンドが見られます。
一日一回、培地量の10%に相当する5%メタノール溶液（水で希釈）を添加し、培地中の0.5%メタノール濃度を維持することが可能です。
効率に違いがある可能性はありますが、一般的に哺乳類由来分泌シグナルは酵母で機能すると考えられます。
可能です。15%ぐりセロールを添加し、-80℃で保存してください。グリセロールストックは凍結融解を繰り返さない限り半永久的に保存できる可能性があります。
グリセロールストックを作成する際、オーバーナイトカルチャーを2-4倍濃縮したものを使用されることをお勧めしています。スピンダウンした細胞を元の25-50%量のグリセロール含有培地に懸濁してください。オーバーナイトカルチャーにぐりセロールを添加するよりも、新鮮な培地にぐりセロールを添加した培地を使用する方が良い結果が得られます。
Bacto-TryptoneとBacto-Peptonは、それぞれ異なるタイプのペプトンです。Bacto-Tryptoneは比較的プアーな窒素源で、多くの窒素はチロシン、トリプトファンから供給されます。Pichia用培地の構成物として両者を比較した場合、増殖曲線はわずかに差が出ます。ただし、ほとんど問題にはなりません。BMGY、BMMYなどPichia培地の中で主な窒素源はYeast Nitrogen Baseですので、両者の影響はほとんどありません。
500 unit Lyticaseは1 unit Zymolaseに相当します。PCR解析の際、1 μLのPichia懸濁液の溶解のためにLyticaseの場合25 unit、Zymolaseの場合は0.05 unit使用されます。また、Zymolaseは精製度の異なるものを入手可能です。全ての用途に20T Zymolaseで十分です。高純度の100T Zymolaseは必ずしも必要ではありません。
pH8～8.5程度まで可能。
次の引用文献では1% casamino acidが使用されています。Clare JJ, et al. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene. 1991 Sep 15;105(2):205-12. PMID: 1937016; UI: 92039033.
本論文で、研究者は以下のように報告しています。PichiaはYP、YNB共に同程度の細胞濃度で生育したが、シングルコピー形質転換体をYNB培地で培養した場合、マウスepidermal growth factor（mEGF）は培地中に非常に低レベルでしか発現しなかった（0.07 μg/mL）。また、培養時間を長くするとさらに発現タンパク質レベルは低下した。一方、pHを6.0に調整し、1% casamino acidを添加したYNBを使用した場合、mEGFレベルは約1.9 μg/mLまで増加した。
通常のPichia発酵条件下（好気的条件）ではエタノールは産生されません。メタノールの酸化的代謝においては、まずホルムアルデヒドが産生され、その後二酸化炭素まで代謝されます。ホルムアルデヒドの同化サイクルも存在しますが、そこでもエタノールは産生されません。
Pichia pastorisは一般的にハプロイド状態で存在します。窒素欠乏下では、交配が起こり、ディプロイドを形成することがあります。同種の細胞同士で交配が起こり得ますので、P. pastorisは定義上雌雄同型です。雌雄異型のSaccharomyces cerevisiaeと比べて、ハプロイド状態のP. pastorisはより安定です。また、窒素欠乏下で、P. pastorisは減数分裂による4つのハプロイド胞子を含む子嚢形成を通してディプロイド化します。
Pichiaのダブリンタイムは約2-3.5時間です（グルコースを添加したSC培地中で）。酵母は一般に30℃でゆっくりと成長します。コロニーが形成されるまでに少なくとも3日を要します。実際に実験に適したコロニーが形成されるのに3-7日間必要です。
必要ありません。Pichia形質転換株はゲノムに発現遺伝子が安定導入されているためです。
Pichia培養液（フラスコ）のpHを調整するためには、5% 水酸化アンモニウム溶液を添加してください。ファーメンターで培養する場合は28%濃縮タイプが使用されます。水酸化アンモニウムはPichia細胞の窒素源としても機能します。
OD600 = 1は5x 10^7 Pichia細胞/mLに相当します。コロニーをピックし、一晩培養した際のPichia培養液のOD600は一般的に1.3-1.5に達します。
Pichiaゲノムは他の酵母のそれと類似しています。ゲノムサイズは1.5 X 10^7（S. cerevisiaeに類似）で、染色体は4本（S. pombeと同様）です。
Yeast 1998 Jul;14(10):895-903 Chromosomal DNA patterns and gene stability of Pichia pastoris. Ohi H, Okazaki N, Uno S, Miura M, Hiramatsu R Osaka Laboratories, Yoshitomi Pharmaceutical Industries Ltd., Japan.
また、我々はcontour-clamped homogeneous electric field gel electrophoresisを用いて、4種のPichia pastoris（Komagataella pastoris）菌株から4種の染色体バンドを分離しました。P. pastoris染色体バンドサイズは、菌株による違いはあったが、1.7 Mb～3.5 Mbの間で、トータルゲノムサイズは9.5 Mb～9.8 Mbと予測されました。
いいえ。Pichia pastoris用ベクターはPichia metahnolicaでは機能しないと考えられます。両者共AOX1プロモーターを持ちますが、相同性が低いため組み換えが起こらないと考えられるためです。また、TEF1プロモーターはPichia methanolicaで機能すると予測されます。
Generally, large colonies represent transformants containing pPIC6/pPIC6α integrants, while small colonies represent transformants containing pPIC6/pPIC6α non-integrants. These non-integrants have transduced the pPIC6/pPIC6α plasmid, and therefore, exhibit a low level of blasticidin resistance in the initial selection process. Upon subsequent screening, these non-integrant transformants do not retain blasticidin resistance.
When choosing a blasticidin-resistant transformant for your expression studies, we recommend that you pick blasticidin-resistant colonies from the initial transformation plate and streak them on a second YPD plate containing the appropriate concentration of blasticidin. Select transformants that remain blasticidin-resistant for further studies.
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Here are some suggestinos:
- Make sure that you have harvested cells during log-phase growth (OD <1.0 generally).
- If electroporation is being used, see the electroporator manual for suggested conditions. Vary electroporation parameters if necessary.
- Use more DNA.
- Use freshly made competent cells.
- If the LiCl transformation method is being used, try boiling the carrier DNA.
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Here are some things to consider:
- If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells.
- Do not use old cells and make sure that they are in log phase of growth.
- Make sure to mix zymolyase well before using. Zymolyase is more of a suspension than a solution.
- Make the PEG solution fresh each time and check the pH.
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Following are the rich and minimal media used for culturing Pichia pastoris and S. cerevisiae:
Rich Media:
S. cerevisiae and Pichia pastoris
YPD (YEPD): yeast extract, peptone, and dextrose
YPDS: yeast extract, peptone, dextrose, and sorbitol
Pichia pastoris only
BMGY: buffered glycerol-complex medium
BMMY: buffered methanol-complex medium
Minimal Media (also known as drop-out media):
S. cerevisiae
SC (SD): Synthetic complete (YNB, dextrose (or raffinose or galactose), and amino acids)
Pichia pastoris
MGY: minimal glycerol medium
MD: minimal dextrose
MM: minimal methanol
BMGH: buffered minimal glycerol
BMMH: buffered minimal methanol
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Use the following high cell density protocol for pGAP clones. Feed carbon until the desired density is reached (300 to 400 g/L wet cell weight (WCW)). If the protein is well-behaved in the fermenter, increase to 300-400 g/L WCW as with methanol inducible clones. These densities can be reached in less than 48 hours of fermentation. We have fermented constitutive expressers on glycerol using these protocols with good results. Some modifications to the Fermentation Basal Salts Medium that you might want to make are:
1) Substitute 2% dextrose for the 4% glycerol in the batch medium.
2) Substitute 40% dextrose for the 50% glycerol in the fed-batch medium.
3) Feed the 40% dextrose at 12 mL/L/hr (Jim Cregg has published data on expression using several carbon sources as substrates; dextrose gave the highest levels of expression).
4) Yeast extract and peptone may be added to the medium for protein stability.
One warning: If you are working with His- strains, they remain His- after transformation with pGAPZ. Fermentation in minimal medium will require addition of histidine to the fermenter.
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No, you cannot autoclave methanol. There are two approaches to this, depending a bit on the size of the bioreactor and the volumes involved. You can either dilute to working concentration and filter-sterilize with a filter suitable for alcohols, or you can just assume that methanol is sterile (it should be) and dilute into sterile water. For the ammonium hydroxide solution, you should also not autoclave it. You can assume the 30% stock solution is sterile (nothing should live in this solution) and dilute into sterile water to the working concentration.
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The use of antibiotics is not recommended, because most antibiotics become inactivated at the low pH of the medium during Pichia fermentation. In other words, addition of antibiotics such as ampicillin or kanamycin won't hurt the fermentation process, but because of the low pH the antibiotics become inactivated or may even precipitate out. For best results, use good sterile techniques.
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You don't have to add sulfuric acid to your PTM1 salts or fermentation medium. It would serve no purpose, other than maybe help dissolve the salts.
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Yes. The cells will do fine in YPD, but there are two drawbacks: The foaming that occurs in the richer YPD is very difficult to control, and the richer medium makes it difficult to purify secreted proteins from the medium. The BMGY formulation remedies both of these problems.
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The use of mixed feeds is mainly due for "turning down" the level of expression for proteins that are troublesome for Pichia. We have generally used mixed feeds for MutS clones. The idea is to keep the culture in a state of more active growth, and thus "happier" to express proteins.
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You need not add any acid to Pichia fermentation media. A healthy culture always acidifies the medium. If the pH of the culture is increasing, it is a sign of carbon source depletion or ill health of the culture.
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It depends whether the clone is Mut+ or a MutS.
For a Mut+ clone, you should expect that initially (in the first 2-4 hours of induction), the oxygen uptake rate of the culture would be lower than that at the end of the glycerol batch phase. After the culture becomes adapted to methanol, the oxygen uptake rate will significantly increase, if the culture is healthy (i.e., not poisoned by too much methanol). One should run methanol spike tests during fermentation of Mut+ clones.
For a MutS clone, one can expect that the oxygen uptake rate will be lower than that at the end of the glycerol batch phase throughout most of the fermentation. One has to be very careful not to poison MutS clones.
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We do not offer any protocols for Pichia fermentation. Please refer to the document titled “Pichia Fermentation Guidelines” on our website.
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The following protocol has been used numerous times for Pichia pastoris. It uses a 250 mL culture that is eventually scaled down to 1 mL aliquots of each strain.
- Inoculate 10 mL YPD media with Pichia strain and grow O/N, shaking at 30 degrees C.
- In the morning, check the OD600. To get them in log phase by the afternoon, dilute cells to hit an OD600 of approximately 3.0 at 4 or 5 pm.
- When the OD600 reaches approximately 3.0, inoculate 250 mL of YPD with 250 µL of culture. The objective is to have healthy, log-phase cells in the morning at an OD600 of around 1.0.
- If the OD600 is ~1.0, spin the cells in a 1 L bottle at 3K rpm for 10 minutes.
- Gently resuspend in 250 mL cold dH20.
- Transfer to a 500 mL centrifuge bottle and spin at 3K for 10 min. Repeat.
- Resuspend in 20 mL cold 1 M sorbitol and transfer to a 50 mL conical tube.
- Spin at 3K rpm for 10 min.
- Resuspend in 1 mL 1M sorbitol, and keep on ice.
- Use 80 µL of host strain for each electroporation.
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Inclusion of 1 M sorbitol in YPD plates stabilizes electroporated cells, as they appear to be somewhat osmotically sensitive.
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PEG 4000 seems to work best for yeast transformations, although PEG 3350 has been used in-house with success.
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We recommend electroporation for transformation of Pichia. Electroporation yields 10e3 to 10e4 transformants per µg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, you may use the Spheroplast Kit for Yeast(Cat. No. K172001), PEG 1000 protocol (page 78 of the manual), LiCl protocol (page 80 of the manual), or the Pichia EasyComp Transformation Kit (Cat. No. K173001). We do not recommend spheroplasting for transformation of Pichia with plasmids containing an antibiotic resistance marker. Damage to the cell wall leads to increased sensitivity to the antibiotic, causing putative transformants to die before they express the antibiotic resistance gene. In contrast, spheroplasting can be used for transformation of PichiaPink vectors because these vectors are selected using auxotrophic markers.
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Here are the different methods available for Pichia transformation:
Pichia EasyComp Transformation Kit: easy-to-use, ready-made reagents
This method produces chemically competent Pichia cells and provides a rapid and convenient alternative to electroporation. Transformation efficiency is low (transformation of 50 µl of competent cells with 3 µg of linearized plasmid DNA yields about 50 colonies), and hence it is very difficult to isolate multi-copy integrants. Higher transformation efficiencies are often obtained with frozen versus freshly prepared cells.
PEG 1000 transformation: easy, do-it-yourself protocol
It is critical to add DNA to frozen cell samples, as cell competence decreases very rapidly after the cells thaw-even when held on ice. To perform multiple transformations, it is recommended to process them in groups of six at a time. The PEG method is usually better than LiCl, but not as good as spheroplasting or electroporation for transformation. However, it is convenient for people who do not have an electroporation device. The transformation efficiency is 10e2 to 10e3 transformants per mg of DNA.
Lithium chloride transformation: easy, do-it-yourself protocol
This method is an alternative to transformation by electroporation. Competent cells must be made fresh. Transformation efficiency is 10e2 to 10e3 transformants per µg linearized DNA. Note: Lithium acetate does not work with Pichia pastoris. Use only lithium chloride.
Electroporation: easy and high efficiency, do-it-yourself protocol; does not destroy the cell wall
Competent cells must be made fresh. Transformation efficiency is 10e3 to 10e4 transformants per µg of linearized DNA.
Spheroplast Kit for Yeast (K172001): cell wall digested to allow DNA to enter the cell; the procedure involves treating cells with zymolyase to create spheroplasts.
You must determine the optimal time to treat with zymolyase by taking OD600 readings at increasing time points. Longer incubations with zymolyase result in reduced transformation efficiency. Spheroplasts are combined with DNA and then plated. Transformation efficiency is 10e3 to 10e4 transformants per µg of linearized DNA.
Note: Spheroplasting is not recommended for Pichia vectors with an antibiotic resistance marker. Damage to the cell wall leads to increased sensitivity to the antibiotic, causing putative transformants to die before they express the antibiotic resistance gene. In contrast, spheroplasting can be used for transformation of PichiaPink vectors, because these vectors are selected using auxotrophic markers.
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Proteinase A is a vacuolar aspartyl protease capable of self-activation, as well as subsequent activation of additional vacuolar proteases, such as carboxypeptidase Y and proteinase B. Carobxypeptidase Y appears to be completely inactive prior to proteinase A-mediated proteolytic processing of the enzyme; proteinase B (encoded by the PrB gene of S. cerevisiae) reportedly is approximately 50% bioactive in its precursor form (i.e., the form that exists prior to proteinase A-mediated processing of the enzyme). Little is known about the proteolytic activities in Pichia pastoris. The following protease-deficient Pichia pastoris strains have been made in an attempt to inactivate or delete the homologous proteolytic activities:
SMD 1168: Pep4 gene disrupted
PichiaPink Strain 2: Pep4 gene disrupted
PichiaPink Strain 3: Prb1 gene disrupted
PichiaPink Strain 4: Prb1, Pep4 genes disrupted
The Pep4-deficient mutant is deficient in protease activity of proteinase A, carboxypeptidase Y, and has approximately one-half of proteinase B activity. The Prb1-deficient mutant is deficient in the activity of proteinase B. Finally, the Pep4/PrB-deficient strain is deficient in proteolytic activity of all three of these enzymes: proteinase A, carboxypeptidase Y, and proteinase B. These protease-deficient strains, when compared to protease wild-type Pichia strains, have been shown to be highly efficient expression systems for the production of proteolytically sensitive products.
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All of our Pichia strains are homothallic strains. This means that they actually switch mating type with each generation. In Saccharomyces strains, this would lead to the culture rapidly becoming entirely diploid. In contrast, Pichia pastoris strains mate inefficiently to form diploids. Therefore, at any given time, the cells in the population are both “a” and “alpha” mating types.
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Multiple strains are provided so you can test which strains work best for a particular protein. It is important to try at least one Mut+ and one MutS strain
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Two genes in Pichia pastoris code for alcohol oxidase-AOX1 and AOX2. The AOX1 gene product accounts for the majority of alcohol oxidase activity in the cell. Expression of the AOX1 gene is tightly regulated and induced by methanol to very high levels. The AOX1 protein typically accounts for about 30% of the total soluble protein in cells grown on methanol. While AOX2 is about 97% homologous to AOX1, growth on methanol is much slower than with AOX1. Loss of the AOX1 gene, and thus a loss of most of the cell's alcohol oxidase activity, results in a strain with a MutS (methanol utilization slow) phenotype. A MutS strain has a mutant aox1 locus, but is wild-type for AOX2. It has reduced ability to metabolize methanol and thus exhibits poor growth on methanol medium. MutS has in the past been referred to as Mut-. Mut+ (methanol utilization plus) refers to the wild-type ability of strains to metabolize methanol as the sole carbon source. Mut+ and MutS phenotypes are used when evaluating Pichia transformants for integration of the gene of interest.
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Pichia pastoris most commonly exists in a vegetative haploid state. Upon nitrogen limitation, mating can occur and diploid cells are formed. Since cells of the same strain can readily mate with each other, P. pastoris is by definition homothallic. Relative to Saccharomyces cerevisiae, which is heterothallic, the haploid state of P. pastoris is more stable. Under nitrogen-limiting conditions, P. pastoris diploids proceed through meiosis to the production of asci containing four haploid spores.
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Upon receipt, we recommend storing Pichia strain stabs at 4 degrees C. For long-term storage, we recommend preparing a glycerol stock (in 15% glycerol) immediately upon receipt and storing at -80 degrees C. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.
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HSA will run at 55 kD on a non-reducing SDS-PAGE gel and at about 66 kDa on a reducing SDS-PAGE gel.
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Yes, you can use the ProBond system with His-tagged proteins expressed in Pichia. Here are some suggestions for using the ProBond system with Pichia supernatant:
- Adjust the pH of the Pichia supernatant to 7.5-8.0.
- Decant the supernatant from the heavy white precipitate. It is recommended to keep the precipitate for later solubilization in the rare case where the expressed protein has co-precipitated.
- Centrifuge the supernatant to remove leftover cell debris or other material that might clog the column.
- Adjust the conductivity to that of 500 mM NaCl with salt addition (may not be required since Pichia media is high salt).
- Run the column according to the instructions in the manual.
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500 units of lyticase must be used to achieve similar effects to 1 unit of zymolyase. To lyse 1 mL of Pichia cells for PCR analysis, 25 total units of lyticase were used compared to 0.05 units of zymolyase. Zymolyase is available in different purities. 20T zymolyase should be sufficient for all purposes. The further purified 100T should not be necessary.
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The lyticase for our protocol is crude lyticase. A pure preparation is more expensive and it is not necessary. We routinely use zymolyase in this protocol for lysing the cells (5 µL of a 1 mg/mL stock). This is excessive, but it works fine. Reference: BioTechniques 20:980-982, June 1996.
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Certain yeast strains secrete a protein toxin, which inhibits the growth of sensitive pathogens and yeasts. Studies have shown that production of the toxin is dependent on the presence of linear, double-stranded DNA plasmids in the killer yeasts. In the yeast Pichia pastoris, two linear double-stranded DNA plasmids have been identified. In the publication listed below, the search for toxin-producing capability in P. pastoris was conducted and no killer activity could be detected when 14 different indicator strains were tested.
Reference: Banerjee and Verma (2000) Search for a Novel Killer Toxin in Yeast Pichia pastoris. Plasmid 43:181-183.
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The molecular weight of the AOX1, and the AOX2 gene product is also 72 kd each.
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No, Pichia pastoris vectors will not work in Pichia methanolica; both Pichia pastoris and Pichia methanolica vectors have promoters derived from alcohol oxidase but they are not homologous, so the Pichia pastoris vectors will not be able to integrate or replicate in Pichia methanolica. The TEF1 promoter is probably functional in Pichia methanolica.
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In the following reference, 1% casamino acids were used: Clare JJ et al. (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105(2):205-212.
In this paper, the researchers found that although Pichia grew to a similar cell density in both YP and YNB, only a very low level of mouse epidermal growth factor (0.07 µg/mL) was present in supernatants from single-copy transformants when grown in YNB, and this decreased during further incubation. By using YNB medium that had been buffered to pH 6.0 and supplemented with 1% casamino acids, secreted mEGF levels substantially increased to ~1.9 µg/mL for single-copy transformants.
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Yeasts in general are known to secrete proteases. There are some proteins specifically susceptible to proteases that have optimal activity at neutral pH. If this is the case, expression using unbuffered media may be indicated. As Pichia expression progresses in an unbuffered medium such as MMH (minimal methanol plus histidine), the pH drops to 3 or below, inactivating many neutral pH proteases. Although the acidic environment of the culture should prevent activity of neutral proteases, you may use PMSF and EDTA at a 1 mM concentration in Pichia crude supernatant (refresh the PMSF every few hours) and then monitor for protease activity. See Deutscher (1990) Guide to Protein Purification, Methods in Enzymology for details.
In contrast, it has been reported that by including 1% Casamino acids (Difco) and buffering the medium at pH 6.0, extracellular proteases were inhibited, increasing the yield of mouse epidermal growth factor. Please see Clare JJ et al. (1991) Gene 105:205-212.
Additionally, major vacuolar proteases may be a factor in degradation, particularly in fermentor cultures that have the combination of the high cell density and lysis of a small percentage of cells. Using a host strain that is defective in these proteases may help reduce degradation. SMD1168 and SMD1168H are protease-deficient Pichia strains that are defective for Pep4p, a proteinase that is required for the activation of other vacuolar proteases, such as carboxypeptidase Y and proteinase B. Please see Higgins DR and Cregg JM (1998) Pichia Protocols, Humana Press, Totowa, New Jersey. Please note that SMD1168, SMD1168H, and the Pichia Protocols book can be ordered from us.
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Under the conditions in which Pichia is fermented (aerobically), ethanol is not produced. Oxidative metabolism of methanol first produces formaldehyde, which is then converted to carbon dioxide. There is an assimilatory cycle involving formaldehyde too, but no ethanol is made in this pathway, either.
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Add 5% ammonium hydroxide solution to maintain the pH of a Pichia culture grown in shake flasks. It is used at a 28% concentrated form in fermentors. Ammonium hydroxide also acts as a nitrogen source for Pichia cells.
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There is no need for maintaining Zeocin antibiotic selection in the Pichia expression medium, since Pichia pastoris transformants are stable integrants with the gene of interest integrated into the genome.
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A secreted protein will be exposed to the glycosylation machinery and might be glycosylated if the protein contains the standard N-linked or O-linked glycosylation amino acid consensus sequence.
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The major advantage of expressing recombinant proteins as secreted proteins is that Pichia pastoris secretes very low levels of native proteins. Since there is a very low amount of protein in the minimal Pichia growth medium, this means that the secreted heterologous protein comprises the vast majority of the total protein in the medium and serves as the first step in purification of the protein.Note: A secreted protein will be exposed to the glycosylation machinery and might be glycosylated if the protein contains the standard N-linked or O-linked glycosylation amino acid consensus sequence.
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Pichia is capable of correctly assembling proteins with a quaternary structure. One of the earliest proteins to be expressed in Pichia was the Hepatitis B Surface antigen which was assembled in its natural form, the 22 nm particle. (Reference: Cregg JM et al. (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast P. Pastoris. Nat Biotechnol 5:479-485.) In consideration of the particle assembly problem, Cregg postulated that one or more post-translational events important in the formation of particles may be slow relative to the synthesis of HBsAg protein. Therefore, he used MutS since it has a slower growth rate.
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You can supplement with 10% culture volume of a 5% methanol (in water) solution to regenerate the 0.5% methanol concentration each day.
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Expression levels are entirely protein-dependent. Please refer to page 60 of the EasySelect manual that lists literature reports for various proteins. The list includes expression levels and type of strain used. The list ranges from 0.001 to 12 grams per liter. Higher expression levels can be achieved with fermentation.
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Zeocin antibiotic can be spread on top of YPD plates for selection of yeast if necessary. There is a report that this works well when done with 10-15 3 mm glass beads. However, it is recommended that some optimization be performed, since top-spreading may dilute the antibiotic's effectiveness.
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The alpha secretion signal is from S. cerevisiae and is a general yeast secretion signal that has been used in many species including P. pastoris, K. lactis, etc.
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The alpha “signal sequence” (which really contains both the alpha signal sequence and pro-hormone leader sequences) is cleaved 4 times by 3 different enzymes in the Pichia cell. First, near the N-terminus by signal peptidase; second, by Kex2p after the dibasic (Lys-Arg) signal slightly upstream of the multiple cloning site, and then twice by Ste13p to remove the 2 Glu-Ala repeats.
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Although the efficiency may differ from one signal to the next, in general mammalian secretion signals are functional in yeast.
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It is doubtful as to whether codon usage plays as great a role in general, as is commonly believed. Translation initiation is probably more of a rate-limiting step than elongation.
Use the following codon usage list to design your gene in the order of preference:
Glycine: GGT or GGA
Glutamic acid: GAG or GAA
Aspartic acid: GAC or GAT
Valine: GTT or GTC
Alanine: GCT or GCC
Arginine: AGA or CGT
Serine: TCT or TCC
Lysine: AAG
Asparagine: AAC
Methionine: ATG
Isoleucine: ATT or ATC
Threonine: ACT or ACC
Tryptophan: TGG
Cysteine: TGT
Tyrosine: TAC
Leucine: TTG or CTG
Phenylalanine: TTC
Glutamine: CAA or CAG
Histidine: CAC or CAT
Proline: CCA or CCT
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An OD600 of 1 is equivalent to 5 x 10e7 Pichia cells/mL. After overnight (O/N) growth from a colony pick, a Pichia culture generally reaches OD 1.3-1.5 (in 2-5 mL).
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Doubling time is 2-3.5 hrs: Pichia has a doubling time of about 2 hrs of glycerol. The yeast grow slowly at 30 degrees C and it takes at least 3 days for colonies. In practice, it takes anywhere from 3 to 7 days to get nice-sized colonies.
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The Pichia genome is similar to that of other yeast, approximately 1.5 x 107 bp (similar to S. cerevisiae) and contains 4 chromosomes (similar to S. pombe). Reference: Ohi H, Okazaki N, Uno S, Miura M, Hiramatsu R (1998) Chromosomal DNA patterns and gene stability of Pichia pastoris. Yeast 14(10):895-903.
We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb, and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains.
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Kex2 cleaves the alpha secretion signal peptide from the N-terminus of the overexpressed protein in yeast. While Ste13 generally removes the Glu-Ala amino acids, which are between the Kex2 site and your experimental protein, the Ste13 cleavage may not be complete. To ensure that the N-terminus of your protein is exposed after Kex2 cleavage, you may clone your gene of interest flush with the Kex2 cleavage site. In order to clone your gene flush with the Kex2 cleavage site, you must cut the pPICalpha vector with XhoI (and any other appropriate enzyme for cloning in-frame with the 3' end of the vector). By cutting the vector with XhoI alone, you will excise 62-67 nucleotides of the vector (depending on which vector you are using), including the Kex2 signal cleavage site. In order to regenerate this signal cleavage site, you will need to generate a forward primer that contains the XhoI site, followed by the Kex2 cleavage sequence and nucleotides from your gene of interest.
After amplification of your gene, you will digest your PCR product with XhoI and, if necessary, with a second restriction enzyme, which will keep your gene of interest in-frame with the C-terminal tag. Please note: The Glu-Ala-Glu-Ala sequence may not be completely essential for cleavage, but it does have an influence. In general, the efficiency of cleavage by the various kexins is affected by sequences both upstream and downstream of the Lys-Arg dibasic motif, but it seems to vary from one member to another. N-terminal sequences may not matter, but amino acid sequences that confer secondary structure that obscures the cleavage site may cause a problem. A number of amino acids are tolerated after the KEX2 cleavage site: these include aromatic amino acids, small amino acids, histidine, and methionine. Proline will inhibit the activity of KEX2.
The EasySelect Pichia Expression Kit allows easy and improved selection with Zeocin antibiotic instead of the HIS marker. The kit also comes with the EasyComp Pichia Transformation Kit instead of the Pichia Spheroplast Kit.
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The EasySelect Pichia Expression Kit features multicopy selection with Zeocin antibiotic selection and can do almost everything the Multi-Copy Pichia Expression Kit can do. The pAO815 vector of the Multi-Copy kit is uniquely suited for in vitro multimerization. The other Multi-Copy vectors have the kanamycin resistance gene that expresses at low level (confers resistance to Geneticin in yeast).
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The P. pastoris expression system combines the benefits of expression in E. coli (high-level expression, easy scale-up, and inexpensive growth) and the advantages of expression in a eukaryotic system (protein processing, folding, and posttranslational modifications), thus allowing high-level production of functionally active recombinant protein. Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. In S. cerevisiae, replicating plasmids are used for episomal expression whereas in Pichia pastoris, plasmids are integrated into the host chromosome.
In comparison to Saccharomyces cerevisiae, Pichia may have an advantage in the glycosylation of secreted proteins because it may not hyperglycosylate. Both Saccharomyces cerevisiae and Pichia pastoris have a majority of N-linked glycosylation of the high-mannose type; however, the length of the oligosaccharide chains added posttranslationally to proteins in Pichia (average 8-14 mannose residues per side chain) is much shorter than those in Saccharomyces cerevisiae (50-150 mannose residues). Very little O-linked glycosylation has been observed in Pichia. In addition, Saccharomyces cerevisiae core oligosaccharides have terminal alpha1,3 glycan linkages whereas Pichia pastoris does not. It is believed that the alpha1,3 glycan linkages in glycosylated proteins produced from Saccharomyces cerevisiae are primarily responsible for the hyper-antigenic nature of these proteins, making them particularly unsuitable for therapeutic use. Although not yet proven, this is predicted to be less of a problem for glycoproteins generated in Pichia pastoris, because it may resemble the glycoprotein structure of higher eukaryotes.
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S. pombe cannot generate P factor when P factor is replaced for alpha in the alpha factor gene. It can, however, produce alpha factor when alpha is replaced for P in the P factor gene. This is negative evidence that S. pombe can process its own mating factor cleavage sites, but not all the cleavage site of the S. cerevisiae alpha factor. It is better to use a more generic signal sequence (rather than a pre- pro- signal sequence such as alpha). If it is necessary to go the pre- pro- route, it is better to use the S. pombe P factor leader, rather than the S. cerevisiae alpha leader.
The use of antibiotics is not recommended because most antibiotics become inactivated at the low pH of the medium during Pichia fermentation. In other words, addition of antibiotics such as Ampicillin or Kanamycin won't hurt the fermentation process, but because of the low pH the antibiotics become inactivated or may even precipitate out. For best results, use good sterile techniques.
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Yes, Pichia should be done under BSL-1 conditions. BSL-1 is the lowest biosafety level.
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GS115/pPICZ/lacZ, GS115/HSA, and GS115 were streaked onto minimal medium containing histidine. Methanol was used as a carbon source. The GS115 and GS115/pPICZ/lacZ grew fast on the methanol plate. The GS115/HSA grew very slowly on methanol plates. The difference in growth rate confirmed that GS115/pPICZ/lacZ is a Mut+ and GS115/HSA is a MutS strain. The addition of fresh histidine to the plates is critical for this experiment to work.
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There is no need for maintaining Zeocin reagent selection in the Pichia expression media since Pichia pastoris transformants are stable integrants with the gene of interest integrated into the genome.
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Zeocin reagent can be spread on top of YPD plates for selection of yeast if necessary. There is a report that this works well when done with 10-15 3mm glass beads. However, it is recommended that some optimization is performed since top-spreading may dilute the antibiotics' effectiveness.