产品信息

The K. lactis Expression Kit provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis (Figure 1). Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC2 (Figure 3). To achieve protein secretion, a gene of interest is cloned downstream of the K. lactis α-mating factor secretion domain (α-MF; Figure 4) which is eventually processed in the Golgi resulting in secretion of the desired protein (Figure 1).

The K. lactis expression system offers several advantages over other yeast and bacterial protein expression systems. First, K. lactis has been used to produce proteins at industrial scale in the food industry for over a decade due to its ability to rapidly achieve high culture densities and abundantly produce recombinant proteins (Figure 2). Second, yeast expression is driven by a variant of the strong LAC4 promoter that has been modified to lack background expression in E. coli (1). Therefore, genes toxic to E. coli can be cloned into pKLAC2 in bacteria prior to their expression in yeast. Third, the kit includes highly competent K. lactis cells making the technology easy-to-use for those not accustomed to working with yeast. Their high transformation efficiency makes the system suitable for methods that require large numbers of transformants, for example, expression cloning using cDNA libraries. Selection of yeast transformants uses a unique antibiotic-free method in which acetamidase (amdS) expressed from pKLAC2 permits transformed cells to utilize acetamide as a sole nitrogen source on defined medium. Acetamide selection promotes formation of cells containing multiple integrations of pKLAC2 which results in higher yields of protein. Finally, proteins expressed in K. lactis have access to eukaryotic protein folding and glycosylation machinery that E. coli cells do not possess, making it an important alternative to bacterial expression systems.

Figure 1:

Figure 2:

Figure 3:

Figure 4:

重点

 

产品类别:

Protein Expression in Yeast Products,

Protein Expression Products

应用:

Protein Expression in Yeast ,

Protein Expression

• 试剂盒组成

  本产品提供以下试剂或组分：

  NEB #	名称	组分货号	储存温度	数量	浓度
  E1000S     Multi-temperature       K. lactis Protein Expression Kit E1000-1 -20       SacII R0157SVIAL -20 1 x 0.1 ml 20,000 units/ml   pKLAC1-malE Control Plasmid N3741SVIAL -20 1 x 0.02 ml 1 mg/ml   pKLAC2 Vector N3742SVIAL -20 1 x 0.02 ml 1 mg/ml   rCutSmart™ Buffer B6004SVIAL -20 1 x 1.25 ml 10 X   Yeast Carbon Base Medium Powder (12 g) B9011SVIAL 4 1 x 12 g Not Applicable   Acetamide solution (sterile) (10 ml) B9018SVIAL 4 1 x 10 ml 100 X   Integration Primer 2 S1278SVIAL -20 1 x 1 ml 50 µg/ml   Integration Primer 1 S1286SVIAL -20 1 x 0.5 ml 50 µg/ml   Integration Primer 3 S1287SVIAL -20 1 x 0.5 ml 50 µg/ml   K. lactis GG799 Competent Cells C1001S -80       K. lactis GG799 Competent Cells C1001SVIAL -80 5 x 0.2 ml Not Applicable   NEB® Yeast Transformation Reagent (5 ml) M2570SVIAL 4 1 x 5 ml Not Applicable

• 特性和用法

  货运单

  • Ships on dry ice

• 优势和特性

  Features

  • High level and scalable expression of recombinant proteins
  • Rapid high cell density growth
  • Simultaneous expression of multiple proteins possible
  • No background gene expression during E.coli cloning steps
  • Easy and fast cell transformation procedure
  • Antibiotic-free selection

• 相关产品

  相关产品

  • BstXI
  • Q5® 热启动超保真 2X 预混液
  • NEB® 5-alpha E. coli 感受态细胞（高效级）
  • NEB® 5-alpha E. coli 感受态细胞（亚克隆效级）

• 注意事项

  1. NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987), NEB 5-alpha Electrocompetent E. coli (NEB #C2989) and NEB 5-alpha Competent E. coli (Subcloning Efficiency) (NEB #C2988) are all recommended for propagation and subcloning pKLAC1 control plasmid and pKLAC2 vector.

• 参考文献

  1. Colussi, P.A. and Taron, C.H. (2005). Appl. Environ. Microbiol.. 71,
  2. Looke, M. et. al (2011). Biotechniques. 50, 325-328.
  3. van Ooyen, A.J.J.et al. (2006). FEMS Yeast Research . 6, 381-392.
  4. Spohner, S.C. et.al (2016). J Biotechnol. 222, 104-116.
  5. Chuzel, L., Ganatra M.B. et. al (2017). Genome Announc. 5, e00623-17.
  6. Read, J.D.et al. (2007). Appl. Environ. Microbiol.. 73, 5088-5096.
  7. Platko, J.D.et al. (2008). Protein Expr. Purif.. 57, 57-62.
  8. Ekborg, N.A.et al. (2007). Appl. Environ. Microbiol.. 73, 7758-7788.
  9. Foster, J.M. et. al (2008). Parasitol Res.. 104(5), 1047.
  10. Vainauskas, S., Taron, C.H. (2016). New England Biolabs Application Note.

操作说明、说明书 & 用法

• 操作说明

  1. Protein expression using the K. lactis Protein Expression Kit – Linearization of pKLAC2 for integrative transformation of K. lactis.
  2. Protein Expression using the K. lactis Protein Expression Kit – Identification of Multi-copy Integrants
  3. Protein Expression using the K. lactis Protein Expression Kit – Identification of properly integrated cells
  4. Protein Expression using the K. lactis Protein Expression Kit – Simultaneous Expression of Multiple Proteins
  5. Protein expression using the K. lactis Protein Expression Kit – Cloning a PCR fragment into pKLAC2 (E1000).
  6. Protein Expression using the K. lactis Protein Expression Kit – Growth of strains for detection of secreted protein
  7. Protein Expression using the K. lactis Protein Expression Kit – Transformation of K. lactis GG799 cells

• 说明书

  产品说明书包含产品使用的详细信息、产品配方和质控分析。

  • manualE1000

• 应用实例

  • Improved method for assembly of linear yeast expression cassettes using NEBuilder HiFi DNA Assembly Master Mix
  • Co-expression of Multiple Proteins in Kluyveromyces lactis

工具 & 资源

• 选择指南

  • Protein Expression and Purification Selection Chart

FAQs & 问题解决指南

• FAQs

  1. What systems does NEB offer for protein expression and purification?
  2. I received my kit and don’t see any competent cells or transformation reagent. Where are they?
  3. I’ve expressed my protein, now how do I purify it?
  4. I’ve noticed untransformed K. lactis will grow when streaked on the selection media. Is the selection not working?
  5. Where can I find more detailed FAQs for the K. lactis Protein Expression Kit?
  6. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

• 实验技巧

  NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520) or the Gibson Assembly Cloning Kit (NEB #E5510) are excellent ways to assemble a linear yeast expression cassette. (See “Improved method for assembly of linear yeast expression cassettes using NEBuilder® HiFi DNA Assembly Master Mix” in the Application Notes section under the Other Tools and Resources tab)