PichiaPink™ Media Kit - FAQs

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74 product FAQs found

我将一个菌落接种到25 mL MGY中，过夜生长，但OD值并不在使用手册中所述的2-6之间。哪里出错了？

这可能是因为接种物较少。我们建议使用起始培养物，这比使用菌落更精确。您可使用倍增时间来计算起始OD。在YPD中，倍增时间约为1.5小时。在MGY中，倍增时间约为3小时。

当使用pPIC6/pPIC6α载体在X-33酵母株中筛选杀稻瘟菌素抗性转化株时，为什么在含300 μg/ml杀稻瘟菌素的YPD培养皿中得到的菌落有大有小？

通常，大菌落代表含pPIC6/pPIC6α整合体的转化株，而小菌落代表含pPIC6/pPIC6α非整合体的转化株。这些非整合体转化株已经转导了pPIC6/pPIC6α质粒，因此，在起始筛选过程中表现出低水平的杀稻瘟菌素抗性。在后续筛选中，这些非整合体转化株不会保持杀稻瘟菌素抗性。

当您为表达实验选择杀稻瘟菌素抗性转化株时，我们建议您从起始转化培养皿中挑选杀稻瘟菌素抗性菌落，然后在第二个含有适当浓度杀稻瘟菌素的YPD培养皿中划线。应选择可保持杀稻瘟菌素抗性的转化株用于下一步研究。

我的转化失败了。你们有何建议？

•应确保收集的是处于对数生长期的细胞（通常OD <1.0）。
•如果使用电穿孔法，应查看电穿孔仪使用手册中的推荐条件。可根据需要，改变电穿孔参数。
•使用更多的DNA。
•使用新鲜配制的感受态细胞。
•如果使用LiCl转化法，可尝试煮沸载体DNA。

我成功制备了两次Pichia的原生质球，之后就制备不成功了。培养物的OD值根本不降低。

应考虑以下几点：

1.如果所用细胞的OD值过高，则它们不能形成原生质球。不要使细胞过度生长。
2.不要使用衰老的细胞，应确保细胞处于对数生长期。
3.使用前，将酵母裂解酶混合均匀。酵母裂解酶更大程度上是一种悬液，而非溶液。
4.每次都使用新鲜配制的PEG溶液，并检查pH。

使用蛋白胨或胰蛋白胨在Pichia培养基中有功能上的差异吗？

细菌用-胰蛋白胨和细菌用-蛋白胨是两种不同的特定类型蛋白胨。细菌用胰蛋白胨是一种稍差的氮源，更多的氮是由酪氨酸和色氨酸提供的。将它们作为Pichia生长培养基的组分进行比较时，生长曲线会稍有不同，但差异很小。在以酵母氮源基础作为主要氮源的Pichia培养基配方中，如BMGY和BMMY，差异非常小或无差异。

1X YNB（酵母氮源基础）的组分和浓度是多少？

在货号为Q30007的包装中，每包所含试剂可制备500 毫升用于Pichia 培养基的10X YNB，或1,000 毫升用于S. cerevisiae培养基的10X YNB（因为Pichia 培养基的用量是S. cerevisiae培养基的2倍）。以下是1X YNB的配方：

硫酸铵 5 g
生物素 0.002 mg
泛酸钙 0.4 mg
叶酸 0.002 mg
肌醇 2 mg
盐酸 0.4 mg
对氨基苯甲酸 0.2 mg
盐酸吡哆辛 0.4 mg
核黄素 0.2 mg
盐酸硫胺素 0.4 mg
硼酸 0.5 mg
硫酸铜 0.4 mg
碘化钾 0.1 mg
六水三氯化铁 0.2 mg
一水硫酸锰 0.4 mg
二水钼酸钠 0.2 mg
一水硫酸锌 0.4 mg
磷酸二氢钾 1 g
硫酸镁 0.5 g
氯化钠 0.1 g
氯化钙 0.1 mg

对YNB培养基（含硫酸铵，无氨基酸）进行灭菌时，能否用高压蒸汽灭菌代替过滤除菌？

可以，含硫酸铵且无氨基酸的YNB培养基可通过高压蒸汽灭菌。

使用组成型表达载体（如pGAPZ）进行发酵，是否有推荐的实验方案？

pGAP克隆可使用以下高细胞密度实验方案。加入碳料，直至达到所需的细胞密度（细胞湿重（WCW）为300-400 克/升）。如果发酵罐中的蛋白状态良好，可增加至300–400 克/升 WCW，与甲醇诱导型克隆相似。在发酵后48小时内，可达到该密度。我们已经使用这些方案，使组成型表达载体在甘油中进行发酵，并得到良好的结果。您可能需要对发酵基础盐培养基做以下改进：

1) 在分批发酵培养基中，用2%葡聚糖代替4%甘油。
2) 在补料生长培养基中，用40%葡聚糖代替50%甘油。
3) 以12 毫升/升/小时的速度补加40%葡聚糖（Jim Cregg已经发表使用多种碳源作为底物进行表达的数据；葡聚糖带来最高表达水平）。
4) 可在培养基中加入酵母提取物和蛋白胨，维持蛋白稳定性。

警告：如果您在使用His-酵母株，使用pGAPZ转化后，酵母株依然是His-标记的。使用基本培养基发酵时，需要在发酵罐中加入组氨酸。

用于制备Pichia发酵培养基的甲醇和氢氧化铵溶液能否进行高压灭菌？

不能，您不能对甲醇进行高压灭菌。有两种方法可解决该问题，一定程度上取决于生物反应器的大小和使用体积。您可将甲醇稀释到工作浓度，并用适用于乙醇的过滤器进行过滤除菌，或者您可以假设甲醇是无菌的（本应该是无菌的）并将其直接稀释到无菌水中。氢氧化铵溶液也不可以进行高压灭菌。您可以假设30%储液是无菌的（没有生物能在该溶液中存活），并使用无菌水将其稀释至工作浓度。

在Pichia发酵期间，是否能够使用抗生素？

不建议使用抗生素，因为在Pichia发酵期间，大部分抗生素会在低pH的培养基中失活。也就是说，加入氨苄青霉素或卡那霉素等抗生素，并不会损害发酵过程，但抗生素会因为低pH条件而失活，甚至沉淀出来。为得到最佳结果，应使用良好的无菌技术。

是否需要在发酵PTM微量盐中加硫酸？

您无需在PTM1微量盐或发酵培养基中加硫酸。这样做除了可能帮助盐溶解，不会产生任何其他作用。

在Pichia发酵中，能否使用YPD培养基代替BMGY培养基？

可以。细胞在YPD中能够良好生长，但存在两个缺点：在营养更丰富的YPD中，很难控制发泡，并且难以从培养基中纯化分泌蛋白。BMGY配方可解决这两个问题。

在Pichia发酵中，使用混合补料的优势是什么？

使用混合补料主要是为了降低对Pichia不利的蛋白的表达水平。我们通常将混合补料用于MutS克隆。目的就是保持培养物处于生长活力旺盛的状态，从而“乐于”表达蛋白。

应使用哪种酸来调节Pichia发酵培养基的pH？是否需要调节pH？

您无需在Pichia发酵培养基中加入任何酸。健康的培养物会使培养基酸化。如果培养物pH在升高，则表示碳源耗尽或培养物不健康。

在Pichia发酵期间，会观察到哪种耗氧模式？

这取决于克隆是Mut+还是MutS。

对于Mut+克隆，起始阶段（诱导的前2-4小时）的培养物耗氧速率低于甘油分批发酵阶段末期。当培养物适应了甲醇后，若培养物健康（即，未被过多的甲醇毒害），则耗氧速率将显著提高。在Mut+克隆发酵期间，应进行甲醇峰值测试。

对于MutS克隆，在发酵过程的大部分时间里，耗氧速率都将低于甘油分批发酵阶段末期。实验人员应非常小心，不要毒害MutS克隆。

你们是否有Pichia发酵实验方案？

我们不提供任何Pichia发酵实验方案。请参考我们网站中名为“Pichia发酵指南”的文档。

对于Pichia细胞的电穿孔实验，是否存在不需要以500 mL细胞为起始的实验方案？

以下实验方案已被多次用于Pichia pastoris。该方案使用250 mL培养物，并最终浓缩至1 mL。

1.将Pichia酵母株接种于10 mL YPD培养基中，30°C震荡过夜生长。
2.第二天早晨，检测OD600。为了使细胞在下午达到对数生长期，应稀释细胞使OD600在下午4点或5点约为3.0。
3.当OD600达到3.0左右，将250 微升培养物接种到250 毫升YPD培养基中。目的是为了在第二天早晨获得健康的对数期细胞，OD600约为1.0。
4.如果OD600约为1.0，则将细胞置于1L的瓶中以3K rpm离心10分钟。
5.轻轻重悬于250 mL预冷的dH20。
6.转移到500 毫升离心瓶中，以3K rpm离心10分钟。重复操作。
7.重悬于20 mL预冷的1 M山梨醇中，并转移至50 mL离心管中。
8.以3K rpm离心10分钟。
9.重悬于1 mL的1M山梨醇中，置于冰上。
10.每次电穿孔使用80 µL宿主酵母株。

在用于接种电穿孔后Pichia细胞的YPD培养皿中加入山梨醇的用途是什么？

在YPD培养皿中加入1 M山梨醇，可稳定经电穿孔的细胞，因为这些细胞对渗透压有些敏感。

是否一定要使用PEG 4000进行酵母转化？

尽管PEG3350已被成功用于内部使用，但PEG 4000的酵母转化效果可能是最好的。

你们建议使用哪种Pichia转化方法？

我们建议使用电穿孔法转化Pichia。采用电穿孔法，每微克线性化DNA可产生103-104个转化株，并且不会破坏Pichia的细胞壁。如果您没有电穿孔设备，则可使用Pichia原生质球试剂盒（货号K172001）、PEG 1000实验方案（使用手册第78页）、LiCl实验方案（使用手册手册第80页）或Pichia EasyComp转化试剂盒（货号K173001）。我们不建议使用原生质球将含有抗生素抗性标记物的质粒转化到Pichia。细胞壁损坏可导致细胞对抗生素的敏感性增强，使假定的转化株在抗生素抗性基因表达前发生死亡。相反，原生质球用可用于PichiaPink载体的转化，因为这些载体是利用营养缺陷型标记物进行筛选的。

Pichia转化有哪些不同的方法？它们之前比较起来有何不同？

以下是不同的Pichia转化方法：

Pichia EasyComp转化试剂盒：易于使用的即用型试剂
该方法可产生化学感受态Pichia细胞，是一种替代电穿孔的快速、方便的方法。转化效率低（使用3 微克线性质粒DNA转化50 微升感受态细胞，得到约50个菌落），因此难以分离多拷贝整合体。与使用新鲜制备的细胞相比，使用冻存细胞可得到更高的转化效率。

PEG 1000转化法：简单、自己动手的实验方案
一定要在冻存细胞样品中加入DNA，因为细胞复苏后，活力降低非常快——即使是在冰上复苏。为了进行多个转化实验，建议分组处理，每次6个。用于转化时，PEG法通常比LiCl更好，但转化效果不如原生质球或电穿孔法。但是，该方法对于不具备电穿孔设备的人来说很方便。转化效率为102-103个转化株/毫克 DNA。

氯化锂转化法：简单、自己动手的实验方案
该方法是电穿孔转化法的替代方法。感受态细胞必须新鲜制备。转化效率为102-103个转化株/微克 线性DNA。

注意：醋酸锂对Pichia pastoris无效。只能使用氯化锂。

电穿孔：简单、高效，自己动手的实验方案；不会破坏细胞壁
感受态细胞必须是新鲜制备的。转化效率为103-104个转化株/微克线性DNA

Pichia原生质球试剂盒：消化细胞壁，使DNA进入细胞；实验步骤包括使用酵母裂解酶处理细胞，从而建立原生质球。
您必须随着时间增加，读取不同时间点的OD600值而确定使用酵母裂解酶进行处理的最佳时间。酵母裂解酶孵育时间过长会导致转化效率降低。将原生质球与DNA结合并接种。转化效率103-104个转化株/微克线性DNA。

注意：不建议将原生质球用于转化带有抗生素抗性标记物的Pichia载体。细胞壁损坏可导致细胞对抗生素的敏感性增强，使假定的转化株在抗生素抗性基因表达前发生死亡。相反，原生质球可用于PichiaPink载体的转化，因为这些载体是利用营养缺陷型标记物进行筛选的。

你们的Pichia酵母株是哪种交配基因型？

我们所有的Pichia酵母株都是同宗配合株。这表示，每一代实际上可转换交配型。在Saccharomyces酵母株中，这会导致培养物迅速完全变为二倍体。相反，Pichia pastoris酵母株不能有效交配形成二倍体。因此，在任何给定的时间内，群体中的细胞既有“a”又有“α”交配型。

Pichia pastoris是否会分泌对自身或其他细胞有毒性的蛋白？

某些酵母株可分泌蛋白毒素，抑制敏感病原体和酵母的生长。研究表明，毒素的产生取决于杀伤酵母中是否有线性双链DNA质粒。在酵母Pichia pastoris中，已鉴定出两种线性双链DNA质粒。下列已发表文章对P. pastoris的毒素生成能力进行了研究，在对14种不同的指示株进行测试时，未检测到杀伤活性。

参考文献：Banerjee and Verma (2000) Search for a Novel Killer Toxin in Yeast Pichia pastoris. Plasmid 43:181­183. 

Pichia pastoris载体（如，pPICZ、pPIC6、pPIC9K、pPIC3.5K和pAO815）在Pichia methanolica中是否有效？TEF1启动子在Pichia methanolica中是否可发挥功能？

不能，Pichia pastoris载体在Pichia methanolica中无效；Pichia pastoris和Pichia methanolica载体都具有来源于乙醇氧化酶的启动子，但不是同源的，因此，Pichia pastoris载体在Pichia methanolica中不能整合或复制。TEF1启动子在Pichia methanolica中可能有效。

当使用含Zeocin抗生素抗性基因的质粒进行Pichia表达时，表达培养基中是否需要含有Zeocin抗生素？

在Pichia表达培养基中，不需要维持Zeocin抗生素筛选，因为Pichia pastoris转化株是稳定的整合载体，已将目的基因稳定整合到基因组中。

在酵母细胞中，重组蛋白经过分泌通路时是否会被糖基化？

分泌蛋白将暴露于宿主细胞的糖基化体系中，如果蛋白质含有标准N-或O糖基化氨基酸共有序列，将可能被糖基化。

使用Pichia培养物进行小规模表达时，出现体积损失，我该怎么办？

您可每天补充10%培养基体积的5%甲醇水溶液，从而再生成0.5%甲醇浓度。

如果没有现成的含Zeocin抗生素的YPD培养皿，是否可以将Zeocin抗生素涂布在YPD培养皿上进行有效的酵母筛选？

如果需要，可将Zeocin抗生素涂布在YPD培养皿上层用于酵母筛选。已有一篇报道称，这种方法与10-15个3 mm玻璃微珠一起使用时非常有效。但是，建议进行一些优化，因为上层挥发会稀释抗生素的效力。

α-因子分泌信号在其他酵母中是否能发挥作用？

α-分泌信号来自S. cerevisiae，是一种通用的酵母分泌信号，已被用于许多种属酵母中，包括P. pastoris和K. lactis等。

α-因子分泌信号序列是如何被加工的？

α“信号序列”（实际上含有α信号序列和激素原前导序列）被Pichia 细胞中3种不同的酶切割4次。首先，信号肽酶在N端附近切割；然后，Kex2p在多克隆位点的二元（Lys-Arg）信号稍上游切割；最后，Ste13p切割两次，除去2个Glu-Ala重复序列。

哺乳类分泌信号在Pichia中是否可以发挥功能？

尽管不同信号的效率可能不同，但是，哺乳类分泌信号通常在酵母中是可以发挥功能的。

Pichia的密码子选择是什么？

密码子选择是否和通常认为的那样具有重要作用是值得怀疑的。翻译起始比延伸更可能成为限速步骤。

使用以下密码子选择清单，按偏好顺序来设计您的基因：

甘氨酸：GGT或GGA
谷氨酸：GAG或GAA
天冬氨酸：GAC或GAT
缬氨酸：GTT或GTC
丙氨酸：GCT或GCC
精氨酸：AGA或CGT
丝氨酸：TCT或TCC
赖氨酸：AAG
天冬氨酸：AAC
甲硫氨酸：ATG
异亮氨酸：ATT或ATC
苏氨酸：ACT或ACC
色氨酸：TGG
半胱氨酸：TGT
酪氨酸：TAC
亮氨酸：TTG或CTG
苯丙氨酸：TTC
谷氨酰胺：CAA或CAG
组氨酸：CAC或CAT
脯氨酸：CCA或CCT

我该如何将OD单位近似转换为Pichia细胞/毫升（或密度）？

OD600值为1相当于5 x 107个Pichia细胞/毫升。挑选出的菌落生长过夜（O/N）后，Pichia培养物通常可达到OD1.3–1.5（在2–5 毫升中）。

Pichia的倍增时间是多久？需要多长时间可在琼脂平板上看到菌落？

在含葡萄糖的SC培养基中，Pichia的倍增时间约为2–3.5小时。酵母在30°C生长缓慢，至少需要3天时间才能长出菌落。实际上，需要3-7天的时间才能够得到大小合适的菌落。

Pichia基因组DNA的大小是多少？

Pichia基因组与其他酵母的相似，约为1.5 x 107 bp（与S. cerevisiae相似），并包含4个染色体（与S. pombe相似）。参考文献：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.

我们利用等强度均电场凝胶电泳，从Pichia pastoris（Komagataella pastoris）酵母株中分离出清晰的4个染色体条带。P. pastoris染色体条带的大小为1.7 -3.5 Mb，总基因组大小预计为9.5-9.8Mb；但是，4个酵母株中的染色体长度呈多态性。

PichiaPink酵母表达系统的筛选机制是什么？

PichiaPink系统利用ADE2互补作用筛选转化株（即腺嘌呤缺陷型的互补）而不是抗生素筛选。ADE2基因可编码磷酸核糖酰氨基眯唑羧化酶，该酶可催化嘌呤核苷酸从头生物合成的第六步。ADE2突变可导致嘌呤前体在液泡中的累积，从而使菌落变成红色。此外，ade2突变体是腺嘌呤营养缺陷型的，在缺乏腺嘌呤的培养基中不能生长，在营养丰富型培养基中生长较慢。

在PichiaPink系统中，由于ADE2基因及其部分启动子完全缺失，菌株为ade2缺陷型。PichiaPink表达载体系统以ADE2基因（由其自身的启动子驱动）为筛选标记物，其高拷贝载体（pPink-HC和pPinkα-HC）含截断的ADE2启动子，而低拷贝载体（pPink-LC）含全长ADE2启动子。使用表达质粒对PichiaPink酵母株进行转化，可使酵母株在缺乏腺嘌呤的培养基上生长（腺嘌呤缺陷型或基本培养基）。无论使用哪种宿主PichiaPink酵母株，使用高拷贝PichiaPink载体进行转化，都能在筛选培养皿上得到白色和淡粉色菌落。菌落颜色间接表明了目的蛋白的相对表达水平，因为菌落颜色取决于质粒拷贝数，而质粒拷贝数取决于标记物的启动子长度。粉色菌落可表达非常少的ADE2基因产物，而白色菌落可表达较多的ADE2基因产物，表明这些菌落具有更高的整合构建体拷贝数。使用低拷贝质粒pPink-LC转化的酵母株，在缺乏腺嘌呤的培养基上生长速度更快，该载体上具有较强的启动子，因而可生成白色克隆。由于具有较强的启动子，较少的ADE2表达就能使酵母株在缺乏腺嘌呤的培养基上生长。因此，酵母株中的ADE2基因/表达构建体拷贝数较低。

PichiaPink酵母表达系统与EasySelect酵母表达系统相比具有哪些优势？

PichiaPink酵母表达系统比传统EasySelect Pichia系统具有显著的优势，具体如下：

-同时具有高拷贝和低拷贝，可优化毒性蛋白表达
-有8个分泌信号前导序列
-4个酵母菌株
-3个蛋白酶缺陷型宿主菌株
-依赖于腺嘌呤筛选而不是抗生素抗性标记

I inoculated 25 mL of MGY with one colony and grew it overnight, but the OD is not 2-6 as mentioned in your manual. Is there something wrong?

This is likely due to low inoculum. We recommend having a starter culture to use, as it is more accurate than using a colony. You can use the doubling time to calculate the starting OD. In YPD, the doubling time is about 1.5 hours. In MGY, the doubling time is going to be about 3 hours.

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When selecting for blasticidin-resistant transformants in the X-33 strain using pPIC6/pPIC6α vectors, why do I get large and small colonies on YPD plates containing 300 µg/ml blasticidin?

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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My transformation is not working. Do you have any suggestions?

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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My spheroplasting of Pichia worked twice, but hasn't worked since. The OD of the culture simply does not drop.

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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Is there a functional difference between using peptone or tryptone for Pichia media?

Bacto-Tryptone and Bacto-Peptone are two different and specific types of peptones. Bacto-Tryptone is a slightly poorer nitrogen source, and more of the nitrogen is provided by tyrosine and tryptophan. When comparing the two as components of media for Pichia growth, growth curves may differ slightly, but there should be only minor differences between the two. In Pichia media formulations that include Yeast Nitrogen Base as a primary source of nitrogen, such as BMGY and BMMY, there should be very little or no difference.

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What are the components and concentrations for 1X YNB (yeast nitrogen base)?

For the pouches sold as Cat. No. Q30007, each pouch contains reagents to prepare 500 mL of 10X YNB for Pichia media or 1,000 mL of 10X YNB for S. cerevisiae media (since Pichia media is 2X what is used for S. cerevisiae). Below is the formulation for 1X YNB:

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
Para-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, 0.4 mg
Potassium iodide, 0.1 mg
Ferric chloride.6H2O, 0.2 mg
Manganese sulfate.H2O, 0.4 mg
Sodium molybdate.2H2O, 0.2 mg
Zinc sulfate.H2O, 0.4 mg
Potassium phosphate monobasic, 1 g
Magnesium sulfate, 0.5 g
Sodium chloride, 0.1 g
Calcium chloride, 0.1 mg

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Can YNB medium (with ammonium sulfate, without amino acids) be autoclaved instead of filter-sterilized?

Yes, YNB medium with ammonium sulfate and without amino acids can be autoclaved for sterilization.

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Is there a recommended protocol for fermentation using constitutive expression vectors such as pGAPZ?

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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Can the methanol and ammonium hydroxide solutions used to prepare Pichia fermentation medium be autoclaved?

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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Can antibiotics be used during Pichia fermentation?

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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Do I need to add sulfuric acid to the fermentation PTM trace salts?

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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Can I use YPD instead of BMGY-type media for Pichia fermentation?

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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What is the advantage of mixed feed in Pichia fermentation?

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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What can be used as an acid to adjust the pH of Pichia fermentation media? Do I even need to adjust the pH?

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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What pattern of oxygen uptake should I expect to observe during a Pichia fermentation run?

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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Do you have any protocols for Pichia fermentation?

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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Is there an electroporation protocol for Pichia cells that doesn't require starting with 500 mL of cells?

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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What is the purpose of including sorbitol in the YPD plates used for plating Pichia cells after electroporation?

Inclusion of 1 M sorbitol in YPD plates stabilizes electroporated cells, as they appear to be somewhat osmotically sensitive.

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Is it critical that one uses PEG 4000 for yeast transformations?

PEG 4000 seems to work best for yeast transformations, although PEG 3350 has been used in-house with success.

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Which method do you recommend using for transformation of Pichia?

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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What are the different methods available for transformation of Pichia, and how do they compare?

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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What is the mating genotype of your Pichia strains?

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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Does Pichia pastoris secrete proteins that can be toxic to itself or to other cells?

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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Will Pichia pastoris vectors (e.g., pPICZ, pPIC6, pPIC9K, pPIC3.5K, pAO815) work in Pichia methanolica? Is the TEF1 promoter functional in Pichia methanolica?

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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When doing Pichia expression from a plasmid containing the Zeocin antibiotic resistance gene, is it necessary to have Zeocin antibiotic in the expression medium?

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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Will a recombinant protein be glycosylated as it goes through the secretory pathway in a yeast cell?

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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What do I do if I see a volume loss during a pilot expression of my Pichia culture?

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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If there are no Zeocin antibiotic-containing YPD plates readily available, would it be possible to spread Zeocin antibiotic on top of YPD plates and still retain efficient selection of yeast?

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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Will the alpha factor secretion signal work in other yeast?

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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How is the alpha factor secretion signal sequence processed?

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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Will a mammalian secretion signal work in Pichia?

Although the efficiency may differ from one signal to the next, in general mammalian secretion signals are functional in yeast.

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What is the codon usage for Pichia?

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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How can I convert OD units to approximate Pichia cells/mL (or density)?

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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What is the doubling time of Pichia? How long should I wait to see colonies on agar?

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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What is the size of the Pichia genomic DNA?

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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What selection mechanism does the PichiaPink Yeast Expression System use?

The PichiaPink system relies on selection of transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. The ADE2 gene encodes phosphoribosylaminoimidazole carboxylase, which catalyzes the sixth step in the de novo biosynthesis of purine nucleotides. Mutations in ADE2 lead to the accumulation of purine precursors in the vacuole, which causes the colony to be red in color. In addition, ade2 mutants are adenine auxotrophs that are unable to grow on medium lacking adenine and have a slow growth phenotype on rich medium.

The strains in the PichiaPink system are ade2 auxotrophs due to the full deletion of the ADE2 gene and part of its promoter. The PichiaPink expression vectors contain the ADE2 gene (under its own promoter) as the selection marker, with the high-copy vectors (pPink-HC and pPinkalpha-HC) containing a truncated ADE2 promoter compared to the full-length ADE2 promoter in the low-copy vector (pPink-LC). Transformation of the PichiaPink strains with the expression plasmids enable the strain to grow on medium lacking adenine (Ade dropout medium or minimal medium). Regardless of the host PichiaPink strain, both white and slightly pink colonies are obtained on the selection plates upon transformation with the high-copy PichiaPink vectors. The color of the colonies indirectly indicates the relative expression levels of the protein of interest as the color of the colony depends on the copy number of the plasmid, which in turn is determined by the promoter strengths of the markers. The pink colonies express very little ADE2 gene product, while the white colonies express higher amounts of the ADE2 gene product, suggesting that those colonies have more copies of the integrated construct. Strains transformed with the low-copy plasmid, pPink-LC, grow faster on medium lacking adenine, generating white colonies due to the stronger promoter on this vector. Since the promoter is stronger, less ADE2 expression is required to allow the strains to grow on medium lacking adenine. As a result, fewer copies of the ADE2 gene/expression construct are required in the strain.

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What are the advantages of the PichiaPink Yeast Expression System over the EasySelect Yeast Expression system?

PichiaPink Yeast Expression System offers significant advantages compared to the original EasySelect Pichia system. Please see the advantages below:

- Both high and low copy enables optimization of toxic protein expression
- 8 secretion signal leader sequences
- 4 strains
- 3 protease-deficienct host strains
- Relies on adenine selection instead of an antibiotic resistance marker

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