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View additional product information for pGAPZ A, B, & C Pichia pastoris Expression Vectors - FAQs (V20020)
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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。
•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。
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。
以下是用于培养Pichia pastoris和S. cerevisia的营养丰富型和基本培养基：
营养丰富型培养基：
适用于S. cerevisiae和Pichia pastoris
•YPD（YEPD）：酵母提取物、蛋白胨和葡聚糖
•YPDS：酵母提取物、蛋白胨、葡聚糖和山梨醇
仅适用于Pichia pastoris
•BMGY：缓冲型甘油复合培养基
•BMMY：缓冲型甲醇复合培养基
基本培养基（又名缺陷型培养基）：
适用于S. cerevisiae
•SC（SD）：完全合成培养基（YNB、葡聚糖（或棉籽糖或半乳糖）以及氨基酸）
适用于Pichia pastoris
•MGY：基本甘油培养基
•MD：基本葡聚糖培养基
•MM：基本甲醇培养基
•BMGH：缓冲型基本甘油培养基
•BMMH：缓冲型基本甲醇培养基
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
蛋白酶A是一种液泡天冬氨酰蛋白酶，可自我激活并随后激活其他的液泡蛋白酶，如羧肽酶Y和蛋白酶B。在蛋白酶A介导的蛋白酶解过程发生之前，羧肽酶Y完全失活；据报道，蛋白酶B（由S. cerevisiae的PrB基因编码）的前体形式（即，在蛋白酶A介导的酶解过程发生前的存在形式）具有50%左右的生物活性。目前，对Pichia pastoris蛋白水解活性的了解很少。为了使酵母株同源蛋白水解活性失活或缺失，制备了蛋白酶缺陷型Pichia pastoris酵母株：
SMD 1168: Pep4基因功能丧失
PichiaPink 酵母株2: Pep4基因功能丧失
PichiaPink 酵母株3: Prb1基因功能丧失
PichiaPink 酵母株4: Prb1, Pep4基因功能丧失
Pep4缺陷型突变缺乏蛋白酶A、羧肽酶Y的蛋白酶活性，约有一半的蛋白酶B活性。Prb1缺陷型突变缺乏蛋白酶B活性。Pep4/PrB缺陷型突变缺乏所有这三种酶的蛋白水解活性：蛋白酶A、羧肽酶Y和蛋白酶B。与蛋白酶野生型Pichia酵母株相比，这些蛋白酶缺陷型酵母株已被证明是生产蛋白水解敏感性产物的高效表达系统。
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中有两个基因编码乙醇氧化酶——AOX1和AOX2。AOX1基因表达产物占据细胞中大部分的乙醇氧化酶活性。AOX1基因表达可被甲醇严格调控，并且诱导后可达到非常高的水平。在甲醇培养基上生长的细胞中，AOX1蛋白通常占可溶性蛋白总量的30%以上。虽然AOX2与AOX1约有97%同源性，但在甲醇培养基上的生长速度比AOX1慢很多。AOX1基因丢失，会导致细胞的大部分乙醇氧化酶活性缺失，从而生成MutS（methanol utilization slow）表型的酵母株。MutS酵母株具有突变的aox1位点，但AOX2是野生型的。MutS酵母株代谢甲醇的能力降低，因此，在甲醇培养基上生长缓慢。MutS过去被称为Mut–。Mut+（methanol utilization plus）表示野生型酵母株具有代谢甲醇并将其作为唯一碳源的能力。Mut+和MutS这两种表型可用来检测目的基因在Pichia转化体中的整合。
Pichia pastoris最常以无性单倍体的形式存在。在氮源限制的条件下，可能出现交配并形成二倍体细胞。由于相同株的细胞易于相互交配，P. pastoris在定义上是同宗配合的。相对于异宗配合的Saccharomyces cerevisiae，P. pastoris的单倍体状态更稳定。在氮源限制的条件下，P. pastoris二倍体可发生减数分裂产生含四个单倍体孢子的子囊。
我们建议在收到后，将Pichia酵母株穿刺培养物保存在4°C。长期保存时，我们建议在收到后立即将其制备成甘油储液（15%甘油）并保存于–80°C。甘油储液可长期保存（除非经过多次冻融）。在制备甘油储液时，我们建议使用过夜培养物并浓缩2-4倍。将细胞离心，并使用25–50%原始体积的甘油/培养基重悬。最好使用新鲜培养基加甘油冻存细胞，而不仅仅是将甘油加到过夜培养物中这么简单。
在非还原型SDS-PAGE凝胶上，HSA将在55 kD；在还原型SDS-PAGE凝胶上，将在66 kD。
可以，您可将ProBond系统用于Pichia表达的His标签蛋白。以下是关于使用ProBond系统纯化Pichia上清液的一些建议：
1.调整Pichia上清液的pH至7.5–8.0。
2.从白色沉淀物中倒出上清液。建议保留沉淀，当表达蛋白发生共沉淀时可将沉淀重新溶解，但这种情况较罕见。
3.离心上清液，除去残留的细胞碎片或其他可能堵塞柱子的物质。
4.加盐，将电导率调整至与500 mM NaCl的电导率相同（可能不需要这样做，因为Pichia培养基是高盐的）。
5.按照使用手册的指示过柱。
500单位的细胞溶解酶与1单位的酵母裂解酶效果相近。为了裂解1 毫升的Pichia细胞用于PCR分析，需使用总共25单位的细胞溶解酶，而酵母裂解酶只需0.05单位。酵母裂解酶有不同纯度。20T酵母裂解酶即能够满足所有用途。没有必要使用纯度更高的100T。
我们的实验方案使用的是粗制细胞溶解酶。纯品较贵，也没有必要使用。在该实验方案中，我们通常使用酵母裂解酶进行细胞裂解（5 微升的1 毫克/毫升储液）。这是过量的，但效果很好。参考文献：BioTechniques 20:980–982, June 1996.
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.
AOX1基因表达产物的分子量为72 kDa（参考文献：Ellis SB, Brust PF, Koutz PJ, Waters AF, Harpold MM, Gingeras TR (1985) Isolation of Alcohol Oxidase and Two other Methanol Regulatable Genes from the Yeast, Pichia pastoris. Mol. Cell. Biol. 5: 1111-1121）。AOX2基因表达产物的分子量也是72 kDa。
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。
在以下参考文献中，使用了1%酪蛋白氨基酸： 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.
在这篇文章中，研究人员发现，尽管Pichia在YP和YNB中的生长密度相近，但在YNB中生长时，单拷贝转化子上清液中只有非常低水平的小鼠表皮生长因子（0.07 微克/毫升），并且该值在后续孵育中继续降低。使用经缓冲处理后pH为6.0的YNB培养基并补充1%酪蛋白氨基酸，单拷贝转化子的mEGF分泌水平大幅增加至1.9 微克/毫升左右。
酵母通常被认为可分泌蛋白酶。有些蛋白对于中性pH条件下具有活性的蛋白酶特别敏感。如果遇到这种情况，可以使用无缓冲型培养基进行表达。随着Pichia在无缓冲型培养基如MMH，（基本甲醇加组氨酸）中表达，pH降低至3或更低，使许多中性pH蛋白酶失活。尽管酸性培养环境会阻碍中性蛋白酶发挥活性，您仍然可在Pichia粗提液中加入浓度为1 mM的PMSF和EDTA（每隔几小时添加新的PMSF），并检测蛋白活性。详见Deutscher (1990)著《酶学方法》中的“蛋白纯化指南”。
相反，也有报道称，通过加入1%酪蛋白氨基酸（Difco）和缓冲液处理使培养基pH为6.0，可抑制细胞外蛋白酶，并提高小鼠表皮生长因子得率。详见Clare JJ et al. (1991) Gene 105:205–212.。
此外，较多的液泡蛋白酶可能是一个降解因素，特别是在含高细胞密度和少量细胞裂解液的发酵培养物中。使用缺乏这些蛋白酶的宿主酵母株，可能有助于减少降解。SMD1168和SMD1168H是蛋白酶缺陷型Pichia株，缺乏Pep4p，Pep4p是激活羧肽酶Y和蛋白酶B等其他液泡蛋白酶所需的一种蛋白酶。详见Higgins DR & Cregg JM (1998), Pichia Protocols, Humana Press, Totowa, New Jersey。请注意，您可从我们这里订购SMD1168、SMD1168H和Pichia Protocols书籍。
Pichia在（有氧）发酵的条件下，不会产生乙醇。甲醇的氧化代谢首先产生甲醛，甲醛随后被转变成二氧化碳。另一种有甲醛参与的同化作用循环，也不会产生乙醇。
加入5%氢氧化铵溶液，维持摇瓶中Pichia培养物的pH。发酵罐中使用28%浓缩形式。氢氧化铵也可作为Pichia细胞的氮源。
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。
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。
将重组蛋白表达为分泌蛋白的主要优势是Pichia pastoris分泌的天然蛋白水平很低。因为在基本Pichia生长培养基中只有非常少量的蛋白质，这表示分泌的外源蛋白构成了培养基中总蛋白的绝大部分，作为蛋白质纯化的第一步。
注意：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。
Pichia能够正确组装具有四级结构的蛋白。乙型肝炎表面抗原是最早在Pichia中表达的蛋白之一，它被组装成其天然形式的22 nm颗粒。（参考文献：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.）考虑到颗粒组装问题，Cregg假设存在一个或多个对于颗粒形成很重要的翻译后事件相对于HbsAg蛋白合成发生较慢。因此，他使用生长速度更慢的MutS。
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-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“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。
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。
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
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）。
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。
我们建议将酵母置于15%甘油中保存在–80°C。甘油储液可长期保存（除非经过多次冻融）。在制备甘油储液时，我们建议使用过夜培养物并将其浓缩2-4倍。将细胞离心，并使用原始体积25–50%的甘油/培养基重悬。最好使用新鲜培养基加甘油冻存细胞，而不仅仅是将甘油加到过夜培养物中这么简单。
P. pastoris表达系统结合了大肠杆菌表达（高水平表达、易于扩大规模和低成本）和真核系统表达（蛋白加工、折叠和翻译后修饰）的优势，从而可使有功能活性的重组蛋白进行高水平生产。Pichia pastoris与Saccharomyces cerevisiae具有相似的分子和基因操作优势，而且其外源蛋白表达水平比Saccharomyces cerevisiae高10-100倍。在S. cerevisiae中，质粒复制进行游离表达；而在Pichia pastoris中，质粒整合到宿主染色体中。
与Saccharomyces cerevisiae相比，Pichia在分泌蛋白的糖基化方面具有优势，因为它可能不会过度糖基化。Saccharomyces cerevisiae和Pichia pastorisN-连接糖基化主要是高甘露糖形式；但是，在Pichia中，翻译后添加到蛋白上的寡糖链长度（平均每条侧链为8-14个甘露糖残基）比Saccharomyces cerevisiae（50-150个甘露糖残基）短很多。在Pichia中观察到的O-连接糖基化非常少。此外，Saccharomyces cerevisiae核心寡糖具有末端α1,3聚糖连接头，而Pichia pastoris没有。人们认为Saccharomyces cerevisiae生产的糖基化蛋白的α1,3聚糖连接头，主要将导致这些蛋白具有高抗原性，使得它们特别不适合于治疗用途。尽管尚未被证明，但推测这一点在Pichia pastoris生产的糖蛋白中不是一个大问题，因为这些糖蛋白可能与更高级的真核糖蛋白结构相似。
我们提供original Pichia pastoris表达系统、PichiaPink表达系统和Saccharomyces cerevisiae酿酒酵母表达系统，用于重组蛋白表达。已知P. pastoris和S. cerevisiae遗传性质已十分明确，均可进行多种翻译后修饰。
P. pastoris毕赤酵母表达系统结合了大肠杆菌表达（高水平表达、易于扩大规模和低成本）和真核系统表达（蛋白加工、折叠和翻译后修饰）的优势，从而可对有功能活性的重组蛋白进行高水平生产。作为一种酵母，毕赤酵母Pichia pastoris与酿酒酵母Saccharomyces cerevisiae具有相似的分子和基因操作优势，而且其外源蛋白表达水平比酿酒酵母Saccharomyces cerevisiae高10-100倍。这些特性使毕赤酵母Pichia pastoris 非常适合用作蛋白表达系统。Pichia表达载体含有强乙醇氧化酶（AOX1）启动子，用于高水平、严格调控的诱导型的表达；或者含有甘油醛-3-磷酸脱氢酶（GAP）启动子，用于高水平的组成型表达。诱导型和组成型表达构建体均整合到P. pastoris基因组，建立蛋白表达水平极高的稳定宿主，特别是在使用发酵器的情况下。我们可提供的Pichia pastoris表达系统包括：
•PichiaPink酵母表达系统：最新的Pichia pastoris表达系统包含低拷贝和高拷贝质粒骨架、8种分泌信号序列和4种酵母菌株，这些有助于优化得到最高的重组蛋白产率。所有PichiaPink载体都含有AOX1启动子，用于高水平诱导型表达；还含有ADE2标记物，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。但是，它们通过不同长度的启动子表达ADE2基因产物，从而决定了整合质粒的拷贝数。在pPink-LC载体中，ADE2标记物的启动子为82 bp，可提供低拷贝表达；在pPink-HC载体，ADE2标记物的启动子为13 bp，可提供高拷贝表达。该系统也可诱导pPinkα-HC载体（含S. cerevisiae α-交配因子前导序列）产生高拷贝数分泌表达，并且还包含8种分泌信号序列可优化分泌表达。
•EasySelect Pichia表达试剂盒：一种传统Pichia 表达试剂盒，包含pPICZ和pPICZα载体，可分别用于目的基因的细胞内和分泌表达。这些载体含有AOX1启动子，可产生高水平的诱导型表达；还含有Zeocin抗生素抗性标记物，可直接进行多拷贝整合载体的筛选。它们有助于对表达的蛋白进行简单的亚克隆、纯化以及快速检测。
•Original Pichia表达试剂盒：该试剂盒包含pPIC9、pPIC3.5、pHIL-D2和pHIL-S1载体，每种载体都含有AOX1启动子，可产生高水平的诱导型表达；还含有HIS4基因，用于在缺乏组氨酸的培养基上筛选his4酵母株。pPIC9带有S. cerevisiae α-因子分泌信号，而pHIL-S1带有Pichia pastoris碱性磷酸酶信号序列（PHO），可指导蛋白质向培养基的转运。pHIL-D2和pPIC3.5专为细胞内表达而设计。
•多拷贝Pichia表达试剂盒：该试剂盒专为最大化表达而设计，包含pPIC3.5K、pPIC9K和pAO815载体，可产生和选择含多个目的基因的Pichia菌株。它们可通过体内方法（pPIC3.5K和pPIC9K）或体外方法（pAO815）分离和生成多拷贝插入片段。所有这些载体都含有AOX1启动子，可产生高水平的诱导型表达；还含有HIS4基因，用于在缺乏组氨酸的培养基上筛选his4菌株。pPIC9K载体可指导表达蛋白的分泌，而从pPIC3.5K和pAO815载体表达的蛋白仍留在细胞内。pPIC9K和pPIC3.5K载体带有卡那霉素抗性标记物，从而使Pichia对Geneticin试剂产生抗性。可通过Geneticin试剂抗性水平高低来鉴定自发的多次插入事件。在缺乏组氨酸的培养基上对Pichia转化株进行筛选，并筛选它们对Geneticin试剂的抗性水平。在高浓度Geneticin试剂中的生长能力，表示多拷贝的卡那霉素抗性基因以及目的基因被整合到了基因组。
为实现在S. cerevisiae中的表达，我们提供pYES 载体系列。每个pYES载体都带有GAL1基因的启动子和增强子序列，可实现诱导型表达。GAL1启动子是使用最广泛的酵母启动子之一，因为它在半乳糖的诱导下具有很强的转录活性。pYES载体还具有2 µ复制起点，可以维持游离的高拷贝数（10-40拷贝/细胞）。
酵母是一种单细胞的真核生物，在成分确定培养基中可快速生长（在含葡萄糖培养基中的倍增时间通常为2.5小时），相比使用昆虫或哺乳细胞生产重组蛋白更简单、更便宜（见下表）。这些良好的特性使酵母适用于从多孔培养板、摇瓶和持续搅拌槽生物反应器到小型试验工厂和工业规模反应器等多种形式的蛋白制备。
实验室最常用的酵母种类是酿酒酵母（Saccharomyces cerevisiae，又名Baker或Brewer酵母）和一些毕赤酵母属（Pichia）的甲醇营养型酵母。S. cerevisiae和P. pastoris的遗传性质均已明确，并能够对蛋白进行翻译后修饰，包括二硫键形成和糖基化，这对于一些重组蛋白发挥正常功能具有重要作用。但是，应注意酵母的糖基化与哺乳细胞的有所不同：在S. cerevisiae中，O-连接的寡糖只有甘露糖残基，而更高等的真核蛋白有唾液酸化的O-连接糖链。此外，已知S. cerevisiae可过度糖基化N-端位点，从而导致蛋白质结合和活性发生改变，并可能在治疗应用中产生异常的免疫原应答。在P. pastoris中，寡糖链的长度短很多，并且已有一个P. pastoris株被报导可产生复杂的、末端唾液酸化的或“人源化”的糖蛋白。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
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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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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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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We recommend storing yeast frozen at -80 degrees C in 15% glycerol. 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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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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We offer the original Pichia pastoris expression systems, PichiaPink expression system, and Saccharomyces cerevisiae yeast expression system for expression of recombinant proteins. Both P. pastoris and S. cerevisiae have been genetically well-characterized and are known to perform many posttranslational modifications.
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. As a yeast, 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. These features make Pichia pastoris very useful as a protein expression system. The Pichia expression vectors contain either the powerful alcohol oxidase (AOX1) promoter for high-level, tightly controlled expression, or the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level, constitutive expression. Both inducible and constitutive expression constructs integrate into the P. pastoris genome, creating a stable host that generates extremely high protein expression levels, particularly when used in a fermentor. The Pichia pastoris expression systems we offer include:
- PichiaPink Yeast Expression System: Newer Pichia pastoris expression system that contains both low- and high-copy plasmid backbones, 8 secretion signal sequences, and 4 yeast strains to help optimize for the highest yield possible of the recombinant protein. All PichiaPink vectors contain the AOX1 promoter for high-level, inducible expression and the ADE2 marker for selecting transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. However, they express the ADE2 gene product from promoters of different lengths, which dictate the copy number of the integrated plasmids. The pPink-LC vector has an 82 bp promoter for the ADE2marker and offers low-copy expression, and the pPink-HC vector has a 13 bp promoter for the ADE2marker and offers high-copy expression. The system also includes the pPinkalpha-HC vector (containing S. cerevisiae alpha-mating factor pre-sequence) for high copy number secreted expression, and provides eight secretion signal sequences for optimization of secreted expression.
- EasySelect Pichia Expression Kit: One of the original Pichia expression kits that contains the pPICZ and pPICZalpha vectors, for intracellular and secreted expression, respectively, of the gene of interest. These vectors contain the AOX1 promoter for high-level, inducible expression and the Zeocin antibiotic resistance marker for direct selection of multi-copy integrants. They facilitate simple subcloning, simple purification, and rapid detection of expressed proteins.
- Original Pichia Expression Kit: The kit includes the pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 vectors, each of which carries the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. pPIC9 carries the S. cerevisiae alpha-factor secretion signal while pHIL-S1 carries the Pichia pastoris alkaline phosphatase signal sequence (PHO) to direct transport of the protein to the medium. pHIL-D2 and pPIC3.5 are designed for intracellular expression.
- Multi-Copy Pichia Expression Kit: This kit is designed to maximize expression and contains the pPIC3.5K, pPIC9K, and pAO815 vectors, which allow production and selection of Pichia strains that contain more than one copy of the gene of interest. They allow isolation and generation of multicopy inserts by in vivo methods (pPIC3.5K and pPIC9K) or in vitro methods (pAO815). All of these vectors contain the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K and pAO815 remain intracellular. The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance marker that confers resistance to Geneticin Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin Reagent. The ability to grow in high concentrations of Geneticin indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome.
- For expression in S. cerevisiae, we offer the pYES Vector Collection. Each pYES vector carries the promoter and enhancer sequences from the GAL1 gene for inducible expression. The GAL1 promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose. pYES vectors also carry the 2m origin and are episomally maintained in high copy numbers (10-40 copies per cell).
Yeast is a single-celled, eukaryotic organism that can grow quickly in defined media (doubling times are typically 2.5 hr in glucose-containing media) and is easier and less expensive to use for recombinant protein production than insect or mammalian cells (see table below). These positive attributes make yeast suitable for use in formats ranging from multi-well plates, shake flasks, and continuously stirred tank bioreactors to pilot plant and industrial-scale reactors.
The most commonly employed species in the laboratory are Saccharomyces cerevisiae (also known as Baker's or Brewer's yeast) and some methylotrophic yeasts of the Pichia genus. Both S. cerevisiae and P. pastoris have been genetically characterized and shown to perform the posttranslational disulphide bond formation and glycosylation that is crucial for the proper functioning of some recombinant proteins. However, it is important to note that yeast glycosylation does differ from that in mammalian cells: in S. cerevisiae, O-linked oligosaccharides contain only mannose moieties, whereas higher eukaryotic proteins have sialylated O-linked chains. Furthermore S. cerevisiae is known to hyperglycosylate N-linked sites, which can result in altered protein binding, activity, and potentially yield an altered immunogenic response in therapeutic applications. In P. pastoris, oligosaccharides are of much shorter chain length and a strain has been reported that can produce complex, terminally sialylated or “humanized” glycoproteins.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
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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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Yes, Pichia should be done under BSL-1 conditions. BSL-1 is the lowest biosafety level.
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Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
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Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.