参见作者原研究论文

本实验方案简略版
May 2020
Advertisement

本文章节


 

Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
在pET表达质粒中实现增加蛋白质产量的新设计   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used modern methods of DNA assembly and directed evolution to identify improved designs for these modules and demonstrated that these designs support higher protein production yields. Herein, we present two PCR protocols for implementing the designs and increasing protein production from existing pET expression plasmids.


Graphic abstract:



A simple workflow for implementing novel designs in pET expression plasmids.


Keywords: pET (pET), Plasmid (质粒), T7lac (T7lac), Transcription initiation (转录起始), Translation initiation region (TIR) (平移起始区(TIR)), Synthetic evolution (合成进化), Bacterial cell factory (细菌细胞工厂), Recombinant protein (重组蛋白)

Background

The basic architecture of pET expression plasmids was established over three decades ago by integrating the ϕ10 promoter for the T7 RNA polymerase (T7p) and the Tϕ transcription terminator (T7t) into the pBR322 backbone (Rosenberg et al., 1987). This architecture enables efficient transcription of cloned coding sequences in bacterial strains harbouring an inducible copy of the DE3 phage fragment encoding the T7 RNA polymerase. The basic pET vector architecture was then elaborated on by including optional add-ons. For example, a Shine-Dalgarno (SD) sequence originating from the major capsid protein of T7 (gene 10 protein) was incorporated to enable efficient translation initiation (Rosenberg et al., 1987), and the lac O1 operator sequence was cloned adjacent to the T7 promoter (T7lac) so that basal gene expression was repressed in the absence of an inducer (Dubendorf and Studier, 1991). Alternative antibiotic cassettes and purification, solubility, and secretion tags were also included. Currently, 103 different pET expression plasmids are available (Shilling et al. 2020). It is also possible to construct bespoke T7p-based expression plasmids using the modular platform for Standard European Vector Architecture (SEVA) (Silva-Rocha et al., 2013).


pET expression plasmids are currently used ‘off-the-shelf’ because they are known to support high titers of recombinant protein production - as much as 50% of the total cell protein after a few hours of induction (Mierendorf et al., 1998). Titers can also be increased by screening induction conditions or testing bacterial hosts that are supplemented with tRNAs and folding catalysts or that modulate the expression of T7 RNA polymerase (Rosano and Ceccarelli, 2014; Rosano et al., 2019). However, not all recombinant proteins can be expressed at high titers and many fall out of experimental pipelines. For example, analysis of structural genomics pipelines indicated that more than 40% of soluble proteins cannot be produced in sufficient titers for downstream structural, biochemical, and biophysical studies (Walsh, 2015; Parret et al., 2016).


In a recent study, we identified a design flaw in the T7lac module (Shilling et al., 2020). This module was originally engineered by fusing T7p to the lac O1 operator sequence in the early generation pET plasmids (Dubendorf and Studier, 1991). T7p is typically 23 nucleotides long and sits −17 to +6 relative to the messenger RNA (mRNA) start site (Figure 1) (Dunn et al., 1983). However, it was truncated by four nucleotides when lac O1 was fused, as the architects used an StuI restriction site within T7p. In our previous study, we used an overlap PCR approach to insert the four truncated nucleotides into T7lac in the most commonly used pET expression plasmid, pET28a(+). We subsequently demonstrated that this design (T7pCONSlac) increased the production titers of recombinant proteins (Shilling et al., 2020). Herein, we present a protocol for incorporating the T7pCONSlac design in vectors with an existing T7lac module (Protocol 1). This protocol is directly applicable to 88 different pET plasmids (Table 1), as well as the +LacIq-PT7/LacO (SEVA#4E) module of the pSEVA platform (Silva-Rocha et al., 2013). The remaining 15 pET vectors encode T7pCONS and do not include the lac O1 operator sequence.



Figure 1. Comparison of T7 promoters. The T7 promoter in pET28a(+) and 87 other pET plasmids is a truncated variant fused to the lac operator. Protocol 1 uses an overlap PCR approach to insert four nucleotides into the T7 promoter, thus restoring the consensus sequence and increasing production titers. Figure adapted from Shilling et al. (2020).


Our recent study also identified a design flaw in the translation initiation region (TIR) of pET28a(+). This module is a stretch of 30 nucleotides that is recognised by the 30S subunit of the ribosome during translation initiation (i.e., the first ribosomal footprint). The sequence determines the efficiency of translation initiation, the rate-limiting step in protein synthesis (McCarthy and Gualerzi, 1990; Laursen et al., 2005; Milón and Rodnina, 2012), and significantly affects the production titers of recombinant proteins (Mirzadeh et al., 2015, 2016 and 2020; Shilling et al., 2020). The sequence is usually comprised of: (1) a Shine-Dalgarno sequence complementary to the 16S rRNA subunit; (2) an AUG start codon that is situated 5-9 nucleotides downstream; and (3) the first 5 codons of the coding sequence (Shine and Dalgarno, 1975; Chen et al., 1994; Osterman et al., 2013). A large body of work indicates that the TIR works most effectively when it is largely free of mRNA structures, which promotes accessibility of the 30S subunit (Kudla et al., 2009; Plotkin and Kudla, 2011; Bentele et al., 2013; Goodman et al., 2013). In most pET expression plasmids, this region is comprised of the SD sequence and a seven-nucleotide spacer region from the major capsid protein of T7, and the first five codons of the coding sequence. However, there is no indication that this region has been optimised in any pET expression plasmid, which we considered a design flaw. We therefore carried out a directed evolution approach on the TIR in pET28a(+), which encodes an N-terminal poly-histidine tag and a thrombin protease cleavage site (Figure 2). We identified two TIRs that work more efficiently than the existing TIR, as judged by the fact that they increase protein production titers (Shilling et al., 2020). Herein, we present a protocol for incorporating the improved TIRs (Protocol 2), which is directly applicable to four of the most widely used pET plasmids (Table 1). Utilising the optimisation strategy, we noted improvements to sfGFP expression levels, starting from a low of 0.8 mg/ml to a high of 97 mg/ml, without affecting protein quality (Shilling et al., 2020). In instances where protein expression was already determined to be high, the addition of optimised TIRs described in this protocol did not always result in an increase in protein yield (unpublished data).



Figure 2. Features of the pET28a(+) plasmid and position of oligonucleotides used in the two PCR protocols. The pET28a plasmid includes the ϕ10 (T7) promoter and the lac operator, as well as the translation initiation region (TIR) encompassing the Shine-Dalgarno (SD) sequence, a spacer, and the first five codons of the open reading frame. Protocol 1 oligonucleotides (blue) incorporate four nucleotides within the T7 promoter at +3-6 relative to the mRNA transcriptional start site. Protocol 2 oligonucleotides (purple) incorporate nucleotide mutations in the TIR, which increases protein production titers.

Materials and Reagents

  1. Petri dishes (VWR, catalog number: 391-0440)

  2. Plate culture spreaders (VWR, catalog number: 612-1561)

  3. Inoculation loops (VWR, catalog number: 612-9352)

  4. 50-ml conical tubes (VWR, catalog number: 525-0402)

  5. Chemically competent E. coli MC1061

  6. Q5 polymerase (NEB, catalog number: M0491L, storage: -20°C)

  7. DpnI (NEB, catalog number: R0176L, storage: -20°C)

  8. dNTPs (Thermo Scientific, catalog number: R0181, storage: -20°C)

  9. Oligonucleotides (Eurofins Genomics)

  10. Agarose (Sigma-Aldrich, catalog number: A9539)

  11. O’GeneRuler DNA Ladder (Thermo Scientific, catalog number: SM1163)

  12. DNA miniprep kit (OmegaTek, catalog number: D6943-02)

  13. Yeast extract (Oxoid, catalog number: LP0021)

  14. Tryptone (Oxoid, catalog number: LP0042)

  15. NaCl (VWR, catalog number: ICNA0219473805)

  16. Agar (VWR, catalog number: 20767.298)

  17. Kanamycin sulfate (VWR, catalog number: 0408-EU-25G, storage: 4°C)

  18. Ampicillin sodium salt (Sigma, catalog number: A0166-25G, storage: 4°C)

  19. LB medium (see Recipes)

  20. Antibiotic stock solutions (see Recipes)

  21. LB agar (see Recipes)

  22. 50× TAE buffer (see Recipes)

  23. DNA loading buffer (see Recipes)

Equipment

  1. Thermocycler (Techne, catalog number: 5PRIME/02)

  2. DNA mini horizontal submarine unit (Hoefer, catalog number: HE33)

  3. Electrophoresis power supply (GE Healthcare, catalog number: EPS601)

  4. Gel Imager Azure C200 (Azure Biosystems, catalog number: AC2001)

  5. Thermomixer Comfort (Eppendorf, catalog number: 5355000.011)

  6. New Brunswick Incubator (New Brunswick, catalog number: M1282-0012)

  7. Benchtop centrifuge (Eppendorf, model: 5417C)

  8. NanoDrop Spectrophotometer (Thermo Scientific, catalog number: ND-2000)

Procedure

  1. Check whether the protocols are relevant to the pET plasmid of interest by cross-referencing Tables 1 and 2. If so, order the required oligonucleotide set.

    1. Protocol 1 will correct a design flaw in the T7lac module, converting it to T7pCONSlac. This protocol only applies to the pET plasmids listed in Table 1.

    2. Protocol 2 will replace the standard TIR with one of two optimised TIRs. This protocol only applies to pET expression plasmids encoding an N-terminal poly-histidine tag, such as those listed in Table 1.


      Table 1. pET plasmids that will benefit from each protocol


    3. Order the oligonucleotide sets required for implementing the T7CONSlac promoter (Protocol 1) and/or the TIR (Protocol 2). The oligonucleotides have complementarity to the plasmid template at their 3’ ends and to each other at their 5’ ends. The sequences are provided in Table 2.


      Table 2. Oligonucleotide sequences for correction of design flaws


  2. PCR setup and program for Protocol 1

    1. Combine the following reagents (Final reaction volume 25 μl):

      1. 5 μl Q5 reaction buffer

      2. 5 μl GC enhancer solution (optional: included with the Q5 polymerase kit)

      3. 1 μl dNTPs from a 10 mM stock (0.4 mM final concentration)

      4. 1.25 μl primer 1 from a 10 μM stock (0.5 μM final concentration)

      5. 1.25 μl primer 2 from a 10 μM stock (0.5 μM final concentration)

      6. 0.5 μl template plasmid from a 2 ng/μl stock (0.04 ng final concentration)

      7. 0.25 μl Q5 polymerase

      8. 10.75 μl sterile ultrapure water

    2. Set up a PCR program with the following parameters:

      1. Hold at 95°C for 5 min

      2. First cycle with 5 repeats

        95°C for 30 s

        48°C for 30 s

        72°C for 3.5 min

      3. Second cycle with 20 repeats

        95°C for 30 s

        60°C for 30 s

        72°C for 3.5 min

      4. Infinite hold at 10°C


  3. PCR setup and program for Protocol 2

    1. Combine the following reagents (TIR-1 or TIR-2) (Final reaction volume 25 μl):

      1. 5 μl Q5 reaction buffer

      2. 5 μl GC enhancer solution (optional)

      3. 1 μl dNTPs from a 10 mM stock (0.4 mM final concentration)

      4. 1.25 μl primer 3 (TIR-1) or 4 from a 10 μM stock (TIR-2) (0.5 μM final concentration)

      5. 1.25 μl primer 5 from a 10 μM stock (0.5 μM final concentration)

      6. 0.5 μl template plasmid from a 2 ng/μl stock (2 ng final concentration)

      7. 0.25 μl Q5 polymerase

      8. 10.75 μl sterile ultrapure water

    2. Set up a PCR program with the following parameters:

      1. Hold at 95°C for 5 min

      2. First cycle with 5 repeats

        95°C for 30 s

        48°C for 30 s

        72°C for 3.5 min

      3. Second cycle with 20 repeats

        95°C for 30 s

        60°C for 30 s

        72°C for 3.5 min

      4. Infinite hold at 10°C


  4. Check the PCR product by agarose gel electrophoresis

    1. Cast a 1% (w/v) agarose gel with 1× TAE buffer and an appropriate gel stain.

    2. Mix 3 μl PCR reaction with a suitable DNA loading buffer.

    3. Load the samples and perform electrophoresis at a constant 100 V for 30 min.

    4. Visualise the gel on a suitable imaging workstation (such as the Azure 200, Azure Biosystems). An example of an expected PCR product is shown in Figure 3.



      Figure 3. Example of an expected PCR product for protocol 2. A 1% agarose gel in TAE buffer. Lane 1: O’GeneRuler 1-kb Plus DNA Ladder (Thermo Scientific). Lane 2: PCR product using primers 3 and 5. Lane 3: PCR product using primers 4 and 5.


  5. Perform DpnI treatment of the PCR product

    1. Combine the following in a 200-μl PCR tube:

      1. 4 μl PCR product (Optional: Perform PCR clean-up of the sample prior to DpnI treatment)

      2. 0.5 μl Cutsmart buffer (included in the DpnI kit)

      3. 0.5 μl DpnI

    2. Incubate the samples at 37°C for 1 h in a thermocycler (such as the Techne Large-Format Gradient Thermal Cycler).

    3. Optional: Heat inactivate DpnI at 80°C for 15 min.


  6. Transform MC1061 with the DpnI-treated PCR product

    1. Add 5 μl DpnI-treated sample to 50 μl chemically competent E. coli such as MC1061 or an alternative strain capable of in vivo DNA assembly.

    2. Incubate on ice for 30 min.

    3. Heat shock at 42°C for 1 min.

    4. Incubate on ice for 2 min.

    5. Add 150 μl LB medium.

    6. Incubate at 37°C for 30 min with shaking at 900 rpm using a thermomixer (such as the Eppendorf Thermomixer R).

    7. Plate 100 μl cell culture onto LB-agar containing a suitable antibiotic.

    8. Allow the plate to dry in an aseptic environment.

    9. Incubate the plate face-down overnight at 37°C.


  7. Pick colonies and sequence

    1. Pick two individually separated colonies and inoculate 10 ml LB media with a suitable antibiotic in a 50-ml conical tube.

    2. Incubate the inoculated cultures at 37°C overnight with shaking at 180 rpm.

    3. Harvest the cultures by centrifugation at 3,220 × g for 10 min.

    4. Extract the plasmids using a miniprep kit such as the E.Z.N.A DNA mini kit from Omega Bio-Tek.

    5. Measure the DNA concentration using a spectrophotometer such as the NanoDrop 2000.

    6. Send the purified plasmids for sequencing.

      Note: Use appropriate primers that are a minimum of 60 bp from the site of mutagenesis.

    7. Confirm insertion of the new modified DNA sequence by comparison with the expected sequence using a standalone program such as SnapGene (download from https://www.snapgene.com/) or an online browser-based program such as Benchling (access from https://www.benchling.com/).

Recipes

  1. LB medium

    10 g/L NaCl

    10 g/L tryptone

    5 g/L yeast extract

    Dissolve in 1 L ultrapure water and sterilise by autoclaving

  2. Antibiotic stock solutions

    Weigh the required quantity of antibiotics; 50 mg/ml kanamycin or 100 mg/ml ampicillin.

    Add ultrapure water to the desired volume and dissolve by vortexing.

    Under aseptic conditions, filter sterilise the antibiotic solution into the appropriate volumes.

    Use directly or store at -20°C.

  3. LB agar

    LB medium plus 15 g/L agar

    Dissolve in 1 L ultrapure water and sterilise by autoclaving.

    Allow the media to cool to ~50°C.

    Add the appropriate antibiotic and pour 20-ml volumes into 9.5-cm Petri dishes.

  4. Tris acetic acid (TAE) (50×)

    242.2 g/L Tris base (2 M)

    57.1 ml/L acetic acid (1 M)

    18.6 g/L EDTA (50 mM)

  5. 6× DNA loading buffer

    60% v/v glycerol

    20 mM Tris pH 8.0

    60 mM EDTA

    0.03% bromophenol blue

Acknowledgments

This work was supported by the Swedish Research Council (2017-00704) and the Carl Tryggers Stiftelse. The original research leading to these protocols is described in Shilling et al. (2020); we would like to acknowledge all co-authors from that publication. We also thank James Cumming and Diana Khananisho for testing the protocols.

Competing interests

The protocols and designs described herein are free from intellectual property. However, the synthetic evolution process used to identify TIR-1 and TIR-2 is patent-protected (PCT/SE2015/051343; European Patent no. 3234146). These patents are the property of CloneOpt AB, of which P.J.S. is a former employee and D.O.D. is a shareholder.

References

  1. Bentele, K., Saffert, P., Rauscher, R., Ignatova, Z. and Blüthgen, N. (2013). Efficient translation initiation dictates codon usage at gene start. Mol Syst Biol 9: 675.
  2. Chen, H., Bjerknes, M., Kumar, R. and Jay, E. (1994). Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res 22(23): 4953-4957.
  3. Dubendorf, J.W. and Studier, F.W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219(1): 45-59.
  4. Dunn, J.J., Studier, F.W. and Gottesman, M. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol 166(4): 477-535.
  5. Goodman, D.B., Church, G.M. and Kosuri, S. (2013). Causes and effects of N-terminal codon bias in bacterial genes. Science 342(6157):475-479.
  6. Kudla, G., Murray, A.W., Tollervey, D. and Plotkin, J.B. (2009). Coding-sequence determinants of gene expression in Escherichia coli. Science 324(5924): 255-258.
  7. Laursen, B.S., Sørensen, H.P., Mortensen, K.K. and Sperling-Petersen, H.U. (2005). Initiation of Protein Synthesis in Bacteria. Microbiol Mol Biol Rev 69(1):101-123.
  8. McCarthy, J.E. and Gualerzi, C. (1990). Translational control of prokaryotic gene expression. Trends Genet 6(3):78-85.
  9. Mierendorf, R.C., Morris, B.B., Hammer, B. and Novy, R.E. (1998). Expression and Purification of Recombinant Proteins Using the pET System. Methods Mol Med 13: 257-292.
  10. Milón, P. and Rodnina, M.V. (2012). Kinetic control of translation initiation in bacteria. Crit Rev Biochem Mol Biol 47(4): 334-348.
  11. Mirzadeh, K., Martínez, V., Toddo, S., Guntur, S., Herrgård, M.J. and Elofsson, A., et al. (2015). Enhanced protein production in Escherichia coli by optimization of cloning scars at the vector-coding sequence junction. ACS Synth Biol 4(9): 959-965.
  12. Mirzadeh, K., Shilling, P.J., Elfageih, R., Cumming, A.J., Cui. H.L. and Rennig. M., et al. (2020). Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microb Cell Fact 19(1): 85.
  13. Mirzadeh, K., Toddo, S., Nørholm, M.H.H. and Daley, D.O. (2016). Codon optimizing for increased membrane protein production: A minimalist approach. Methods Mol Biol1432: 53-61.
  14. Osterman, I.A., Evfratov, S.A., Sergiev, P.V. and Dontsova, O.A. (2013). Comparison of mRNA features affecting translation initiation and reinitiation. Nucleic Acids Res 41(1): 474-486.
  15. Parret, A.H., Besir, H. and Meijers, R. (2016). Critical reflections on synthetic gene design for recombinant protein expression. Curr Opin Struct Biol 38:155-162.
  16. Plotkin, J.B. and Kudla, G. (2011). Synonymous but not the same: the causes and consequences of codon bias. Nat Rev Genet 12(1): 32-42.
  17. Rosano, G.L. and Ceccarelli, E.A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172.
  18. Rosano, G.L., Morales, E.S. and Ceccarelli, E.A. (2019). New tools for recombinant protein production in Escherichia coli : A 5-year update. Protein Sci 28(8):1412–1422.
  19. Rosenberg, A.H., Lade, B.N., Dao-shan, C., Lin, S.W., Dunn, J.J., Studier, F.W. (1987). Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56(1): 125-135.
  20. Shilling, P.J., Mirzadeh, K., Cumming, A.J., Widesheim, M., Köck, Z. and Daley, D.O. (2020). Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol 3(1): 214.
  21. Shine, J. and Dalgarno, L. (1975). Determinant of cistron specificity in bacterial ribosomes. Nature 254(5495): 34-38.
  22. Silva-Rocha, R., Martínez-García, E., Calles, B., Chavarría, M., Arce-Rodríguez, A. and de las Heras, A., et al. (2013). The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Research 41(D1): D666-D675.
  23. Walsh, G. Large-Scale Protein Production. In: Proteins: Biochemistry and Biotechnology, Second Edition. 141-176.

简介

[摘要] pET表达质粒被广泛用于生物技术,生物制药,和基础研究领域用于生产重组蛋白。典型地,它们被用于关闭的,现成的,因为它们支持高PROD ,减税蒂特ř小号; ^ h H但是,我们在许多宠物质粒,限制其生产能力的确定了两个设计缺陷。我们使用现代 DNA 组装方法和定向进化来确定这些模块的改进设计,并证明这些设计支持更高的蛋白质产量。在此,我们提出了两种 PCR 方案,用于实施设计和增加现有pET 表达质粒的蛋白质产量。

图文摘要:

在 pET 表达质粒中实施新颖设计的简单工作流程。


[背景] pET 表达质粒的基本结构是在三十多年前通过将 T7 RNA 聚合酶 ( T7p )的 φ10 启动子和 Tφ 转录终止子 ( T7t ) 整合到 pBR322 骨架中而建立的(Rosenberg等,1987)。此结构使小号在细菌菌株携带的诱导型复制克隆编码序列的有效转录DE3编码T7 RNA聚合酶的噬菌体片段。然后通过包含可选的附加组件详细阐述了基本的 pET 载体架构。例如,一个服务-从T7(基因10蛋白)的主要衣壳蛋白Dalgarno(SD)序列始发掺入以使有效的翻译起始(罗森堡等人。,1987) ,以及LAC Ò 1操纵子序列克隆相邻于T7启动子(T7lac启动),以使得基础基因表达在不存在抑制的诱导(迪本多夫和Studier ,1991) 。替代抗生素磁带和净化,溶解度,也包括和分泌标签。目前,有 103 种不同的 pET 表达质粒可用(Shilling等,2020)。还可以使用标准欧洲载体体系结构 (SEVA) 的模块化平台构建定制的基于 T7p 的表达质粒(Silva-Rocha等,2013)。

pET 表达质粒目前是“现成的”使用,因为已知它们支持高滴度的重组蛋白生产;之多总细胞蛋白的50%诱导后几个小时(Mierendorf等人。,1998) 。蒂特- [R S可也通过筛选诱导条件或测试被补充有tRNA和折叠催化剂或调节细菌宿主增加的expressi上T7 RNA聚合酶(Rosano和切卡雷利,2014; Rosano等人。,2019 )。然而,并非所有重组蛋白都能以高滴度表达,并且许多重组蛋白不符合实验流程。例如,结构基因组学管道的分析表明可溶性蛋白的多于40%的C的ñ ö吨足够滴度来生产ř小号为下游结构,生化,和生物物理研究(沃尔什,2015; Parret等人。,2016).

在最近的研究中,我们发现了一个设计缺陷T7lac启动模块(先令等人。,2020) 。该模块最初是由熔合工程T7p到所述LAC Ò 1在早代pET质粒操纵基因序列(迪本多夫和Studier ,1991) 。T7p通常是23个核苷酸长,-17至6相对于所述信使RNA坐在(mRNA)的开始点(图1) (邓恩等人。,1983) 。然而,当lac O 1融合时,它被四个核苷酸截断,因为设计者在T7p 中使用了Stu I 限制位点。在我们之前的研究中,我们使用重叠 PCR 方法将四个截短的核苷酸插入到最常用的 pET 表达质粒 pET28a(+) 中的T7lac中。我们随后证明这种设计(T7p CONS升AC )增加的生产滴度ř小号的重组蛋白(先令等人。,2020) 。在这里,我们提出了一个协议,用于将T7p CONS lac设计与现有的T7lac模块(协议 1)结合到载体中。此协议是直接适用于88个不同pET质粒(表1),以及在+ LacIq-PT7 / LACO的pSEVA平台(SEVA#4E)模块(Silva的-罗沙等人。,2013) 。其余 15 个 pET 载体编码T7p CONS ,不包括lac O 1操作序列。

图 1. T7启动子的比较。pET28a(+) 和 87 个其他 pET 质粒中的T7启动子是与lac 操纵基因融合的截短变体。协议1个使用重叠PCR方法以插入四个核苷酸中的T7启动子,从而恢复共有序列和增加生产滴度ř秒。图改编自Shilling等人。( 2020) 。

我们最近的研究还发现了pET28a(+)翻译起始区 ( TIR )的设计缺陷。该模块是30个核苷酸的recogni拉伸小号ED由翻译起始期间30S亚基核糖体的(即。,第一核糖体足迹)。顺序决定翻译的效率开始,在蛋白质合成中的限速步骤(McCarthy和Gualerzi ,1990;劳森等人。,2005;米隆和Rodnina ,2012) ,和显著影响的重组蛋白的生产滴度(Mirzadeh等。人,2015年,2016和2020;先令等人。,2020) 。该序列通常包括:(1) 与 16S rRNA 亚基互补的 Shine-Dalgarno 序列;(2) 位于下游 5-9 个核苷酸的 AUG 起始密码子;(3) 编码序列的前 5 个密码子(Shine and Dalgarno , 1975; Chen et al . , 1994; Osterman et al . , 2013) 。大机构的工作表示小号的TIR工作最有效的,当它在很大程度上是免费的mRNA结构,这促进了30S亚单位的无障碍(的Kudla等人。,2009;普洛特金及的Kudla ,2011; Bentele等人。,2013;古德曼等人。,2013) 。在大多数 pET 表达质粒中,该区域由SD 序列和来自 T7 主要衣壳蛋白的七核苷酸间隔区以及编码序列的前五个密码子组成。然而,没有迹象表明该区域已在任何 pET 表达质粒中进行了优化,我们认为这是一个设计缺陷。因此,我们进行了定向进化方法的TIR中的pET28a(+),其编码的N端聚- ħ istidine标签和凝血酶的蛋白酶切割位点(图2)。我们确定了两个TIRS的工作比现有的更有效的TIR ,作为判断的事实,他们增加蛋白质的生产滴度[R小号(先令等人。,2020) 。在此,我们提出了一种用于纳入改进的TIR (协议 2)的协议,该协议直接适用于四种最广泛使用的 pET 质粒(表 1)。Utili š荷兰国际集团的优化解小号通货膨胀策略,我们注意到从0.8毫克/低开始改进的sfGFP表达水平米升至97毫克/高米升,在不影响蛋白质量(先令等人。,2020) 。在已确定为高的蛋白表达的情况下,所述另外的优化解小号编TIRS在这个协议描述并不总是导致增加的蛋白质产率(未公布的数据)。

图 2. pET28a(+) 质粒的特征和两种 PCR 方案中使用的寡核苷酸的位置。所述的pET28a质粒包括φ10(T7)启动子和lac操纵子,以及翻译起始区(TIR包围服务)- Dalgarno(SD)序列,间隔,和所述开放读码框的前五个密码子。方案 1 寡核苷酸(蓝色)在相对于 mRNA 转录起始位点的 +3-6 处的 T7 启动子中加入了四个核苷酸。协议2个寡核苷酸(紫色)把核苷酸突变中的TIR ,这增加小号蛋白质生产滴度ř秒。

关键字:pET, 质粒, T7lac, 转录起始, 平移起始区(TIR), 合成进化, 细菌细胞工厂, 重组蛋白

材料和试剂
 
1.培养皿(VWR,目录号:391-0440)      
2.平板培养摊铺机(VWR,目录号:612-1561)      
3.接种环小号(VWR,目录号:612-9352)      
4. 50 -毫升锥形管小号(VWR,目录号:525-0402)      
5.化学感受态大肠杆菌MC1061      
6. Q5聚合酶(NEB,目录号:M0491L,储存:-20°C)      
7. Dpn I(NEB,目录号:R0176L,储存:-20 °C )      
8. dNTPs(Thermo Scientific,目录号:R0181,储存:-20 °C )      
9.寡核苷酸(Eurofins Genomics)      
10.琼脂糖(Sigma-Aldrich,目录号:A9539)   
11. O'GeneRuler DNA Ladder(Thermo Scientific,目录号:SM1163)   
12. DNA小量制备试剂盒(OmegaTek,目录号:D6943-02)   
13.酵母提取物(Oxoid,目录号:LP0021)   
14.胰蛋白胨(Oxoid,目录号:LP0042)   
15. NaCl(VWR,目录号:ICNA0219473805)   
16.琼脂(VWR,目录号:20767.298)   
17.硫酸卡那霉素(VWR,目录号:0408-EU-25G,储存:4 °C )   
18.氨苄西林钠盐(Sigma,目录号:A0166-25G,储存:4 °C )   
19. LB培养基(见ř ecipes)   
20.抗生素原液(见配方)   
21. LB琼脂(参见ř ecipes)   
22. 50 × TAE缓冲液(见ř ecipes)   
23. DNA上样缓冲液(见ř ecipes )   
 
设备
 
热循环仪(Techne,目录号:5PRIME/02)
DNA微型水平潜艇单元(Hoefer,目录号:HE33)
电泳p奥尔供给(GE ħ ealthcare,目录号:EPS601)
凝胶I mager Azure C200(Azure Biosystems,目录号:AC2001)
Thermomixer中Ç omfort仪(Eppendorf ,目录号:5355000.011)
新不伦瑞克我ncubator(新不伦瑞克省,目录号:M1282-0012)
台式离心机(Eppendorf ,型号:5417C)
NanoDrop S分光光度计(Thermo Scientific ,目录号:ND-2000)
 
程序
 
检查协议是否是相关的宠物通过交叉参考表1和表2。如果是这样的质粒的兴趣,订单的要求寡核苷酸组。
协议 1 将纠正 T7 lac模块中的设计缺陷,将其转换为T7p CONS lac。该协议仅适用于表 1 中列出的pET 质粒。
协议 2 将用两个优化的TIR 之一替换标准 TIR 。该协议仅适用于编码N端聚- pET表达质粒ħ istidine标签,例如那些在表1中列出。
表 1.将受益于每个协议的pET 质粒
 
订购实施 T7 CONS lac启动子(协议 1)和/或 TIR(协议 2)所需的寡核苷酸集。寡核苷酸在其 3' 端与质粒模板互补,在 5' 端彼此互补。序列在表 2 中提供。
 
表 2.用于纠正设计缺陷的寡核苷酸序列
 
协议 1 的 PCR 设置和程序
结合以下试剂(最终反应体积 25 μl):
5 μl Q5 反应缓冲液
5 μl GC 增强剂溶液(可选:包含在 Q5 聚合酶试剂盒中)
1 μl dNTP,来自 10 mM 储备液(0.4 mM 终浓度)
10 μM 原液中的 1.25 μl 引物 1(0.5 μM 终浓度)
1.25 μl 引物 2 来自 10 μM 原液(0.5 μM 终浓度)
0.5 μl 模板质粒来自 2 ng/ μ l原液(0.04 ng 终浓度)
0.25 μl Q5 聚合酶
10.75 μl 无菌超纯水
使用以下参数设置 PCR 程序:
在 95°C 下保持 5 分钟
第一个循环,重复 5 次
95°C 30秒
48°C 30秒
72°C 3.5 分钟
第二个循环,重复 20 次
95°C 30秒
60°C 30秒
72°C 3.5 分钟
10°C 无限保温
 
协议 2 的 PCR 设置和程序
结合以下试剂(TIR-1或TIR-2 )(最终反应体积 25 μl):
5 μl Q5 反应缓冲液
5 μl GC 增强剂溶液(可选)
1 μl dNTP,来自 10 mM 储备液(0.4 mM 终浓度)
1.25 μl 引物 3 (TIR-1) 或 4来自 10 μM 原液(TIR-2)(0.5 μM 终浓度)
10 μM 原液中的1.25 μl 引物 5 (0.5 μM 终浓度)
0.5 μl 模板质粒来自 2 ng/μ l原液(2 ng 终浓度)
0.25 μl Q5 聚合酶
10.75 μl 无菌超纯水
使用以下参数设置 PCR 程序:
在 95°C 下保持 5 分钟
第一个循环,重复 5 次
95°C 30秒
48°C 30秒
72°C 3.5 分钟
第二个循环,重复 20 次
95°C 30秒
60°C 30秒
72°C 3.5 分钟
10°C 无限保温
 
检查所述用琼脂糖凝胶电泳PCR产物
用 1 × TAE 缓冲液和适当的凝胶染色剂浇铸 1% (w/v) 琼脂糖凝胶。
拌3 μ升与PCR反应一个合适的DNA加样缓冲液。
加载的样品和在恒定100伏电泳进行30分钟。
在合适的成像工作站(如 Azure 200、Azure Biosystems)上可视化凝胶。图 3 显示了预期 PCR 产物的示例。
 
 
图 3.方案 2的预期 PCR 产物示例。TAE 缓冲液中的 1% 琼脂糖凝胶。泳道 1:O'GeneRuler 1 - kb Plus DNA Ladder(Thermo Scientific)。泳道 2:使用引物 3 和 5 的 PCR 产物。泳道 3:使用引物 4 和 5 的 PCR 产物。
 
对PCR 产物进行Dpn I 处理
结合在200以下-微升PCR管:
4微升的PCR产物(可选:执行PCR清理的样品之前的DpnI予处理)
0.5 μl Cutsmart 缓冲液(包含在Dpn I套件中)
0.5 微升Dpn I
孵育的样品在37℃下为1个在热循环(如Techne公司大幅面梯度热循环仪)小时。
可选:在 80°C 下加热灭活Dpn I 15 分钟。
 
变换MC1061用的DpnI我-处理PCR产物
加入5微升的DpnI我-处理过的样品至50μl化学感受态大肠杆菌(E.coli) ,如MC1061或一个能够替代应变体内DNA装配。
在冰上孵育 30 分钟。
热休克在42℃下为1分钟。
在冰上孵育2分钟。
添加 150 μl LB 培养基。
使用热混合器(例如 Eppendorf Thermomixer R)以 900 rpm摇动在 37°C 下孵育30 分钟。
板100微升细胞培养物到LB-一个GAR含有一个合适的抗生素。
允许该板在无菌环境中干燥。
在 37°C下将板面朝下孵育过夜。
 
选择菌落和序列
选择两个单独分开的菌落,接种10毫升LB培养基用一个合适的抗生素,在50 -毫升锥形管中。
将接种的培养物在 37°C 下孵育过夜,并以 180 rpm 的速度摇晃。
收获的通过离心培养物在3 ,220 ×克10分钟。
提取的使用小量制备试剂盒,例如自Omega酶标仪的EZNA DNA mini试剂盒的质粒。
测量的使用分光光度计DNA浓度,如纳米滴2000(Thermo Scientific)进行。
发送的纯化质粒进行测序。
注意:使用适当的引物是一个微量庵从突变的网站60基点。
新的修饰的DNA序列插入确认通过比较与预期序列使用一个独立的程序,例如SnapGene (下载从https://www.snapgene.com/)或在线浏览器-基于程序如从Benchling(接入HTTPS:/ /www.benchling.com/)。
 
食谱
 
LB培养基
10 克/升氯化钠
10g / L的吨ryptone
5 g/L y东方提取物
溶解在1大号ü ltrapure水和通过高压灭菌消毒
一个ntibiotic原液
权衡的抗生素的需要的量S; 50毫克/米升ķ anamycin或100mg /米升一mpicillin
加入超纯水的所需量和涡旋溶解
在无菌条件下,过滤器消毒的抗生素溶液进入的适当体积
直接使用或-20°C保存
LB琼脂
LB 培养基加 15 g/L 琼脂
溶解在1大号ü ltrapure笏ER和通过高压灭菌消毒
让介质冷却至 ~50°C
甲DD合适的抗生素和倾20 -米升体积成9.5 -厘米的培养皿
三醋酸 (TAE) (50 × )
242.2 g/L Tris 碱 (2 M)
57.1 毫升/升醋酸 (1 M)
18.6 克/升 EDTA (50 mM)
6 × DNA 上样缓冲液
60% v/v 甘油
20 mM Tris pH 8.0
60 mM EDTA
0.03% 溴酚蓝
 
致谢
 
钍是工作是由瑞典研究理事会的支持(2017-00704 ),并在卡尔Trygger小号小号tiftelse。最初的研究导致这些协议我S中描述先令等。( 2020) ; 我们要感谢该出版物的所有合著者。我们还感谢 James Cumming 和 Diana Khananisho 测试协议。
 
利益争夺
 
这些协议和设计这里描述的是从知识产权的自由。然而,合成的演变过程用于识别TIR-1和TIR-2是专利-保护的(PCT / SE2015 / 051343;欧洲专利没有3234146)。这些专利是 CloneOpt AB 的财产,其中 PJS 是前雇员,DOD 是股东。
 
参考
 
本特尔,K 。,萨弗特,P 。,劳舍尔,R 。,伊格纳托娃,Z 。和Blüthgen , N. (2013)。有效的翻译起始决定了基因开始时的密码子使用。分子系统生物学9:675。
陈,H 。,比约克内斯,M 。,库马尔,R 。和Jay , E. (1994)。确定 Shine-Dalgarno 序列与大肠杆菌 mRNA 的翻译起始密码子之间的最佳比对间距。核酸研究22(23): 4953 - 495 7.
杜本多夫,J 。w ^ 。和学生,F 。W. (1991)。通过用 lac 阻遏物阻断目标 T7 启动子来控制诱导型 T7 表达系统中的基础表达。J Mol Biol 219(1): 45 - 59。
邓恩,J 。Ĵ 。,学生,F 。w ^ 。和Gottesman , M. (1983)。噬菌体 T7 DNA 的完整核苷酸序列和 T7 遗传元件的位置。J Mol Biol 166(4): 477 - 535。
古德曼,D 。乙。,教堂,G 。米。和Kosuri , S. ( 2013 )。细菌基因中 N 端密码子偏倚的原因和影响。科学342(6157):475 -47 9.
库德拉,G 。,默里,A 。w ^ 。,托勒维,D 。和普洛特金,J 。B. (2009 年)。大肠杆菌基因表达的编码序列决定因素。科学324(5924):255 -25 8。
劳尔森,B 。小号。,索伦森,H 。P 。,莫滕森,K 。克。和斯珀林-彼得森,H 。美国(2005 年)。细菌中蛋白质合成的启动。Microbiol Mol Biol Rev 69(1):101 -1 23。
麦卡锡,J 。乙。和Gualerzi , C. (1990)。原核基因表达的翻译控制。趋势基因6(3):78 - 85。
米伦多夫,R 。Ç 。,莫里斯,B 。乙。,锤子,B 。和诺维,R 。E. (1998)。使用 pET 系统表达和纯化重组蛋白。分子医学方法13:257 - 2 92。
米隆,P 。和罗德尼娜,M 。五(2012)。细菌翻译起始的动力学控制。Crit Rev Biochem Mol Biol 47(4): 334 -3 48。
米尔扎德,K 。,马丁内斯,V 。,托多,S 。,冈图尔,S 。,赫尔加德,M 。Ĵ 。和埃洛夫森,A 。,等人。(2015)。增强的p rotein p在roduction大肠杆菌通过Ó的ptimization Ç loning小号在汽车v ector- Ç oding小号层序Ĵ Ĵ结。ACS 合成生物学4(9):959 -9 65。
米尔扎德,K 。,先令,P 。Ĵ 。, Elfageih , R . ,卡明,A 。Ĵ 。,崔。H . 升。和雷尼格。米。,等。(2020)。通过翻译起始区的定向进化增加大肠杆菌中周质蛋白的产量。微生物细胞事实19(1): 85。
米尔扎德,K 。,托多,S 。,诺尔霍尔姆,M 。H . H . 和戴利,D 。O. (2016)。密码子ø ptimizing用于我ncreased米embrane p rotein p roduction:阿米inimalist一个接近角。方法 Mol Biol 1432: 53 - 61。
奥斯特曼,我。一个。,叶夫拉托夫,S 。一个。,谢尔盖耶夫,P 。伏。和Dontsova ,O 。答(2013 年)。影响翻译起始和再起始的 mRNA 特征的比较。核酸研究41(1): 474 -4 86。
帕雷特,A 。H . ,贝西尔,H 。和梅杰斯,R. (2016 年)。重组蛋白表达合成基因设计的批判性思考。Curr Opin Struct Biol 38:155 -1 62。
普洛特金,J 。乙。和Kudla , G. (2011)。同义但不相同:密码子偏倚的原因和后果。Nat Rev Genet 12(1): 32 - 42。
罗萨诺,G 。升。和切卡雷利,E 。答(2014 年)。大肠杆菌中的重组蛋白表达:进展与挑战。前微生物5:172。
罗萨诺,G 。升。,莫拉莱斯,E 。小号。和切卡雷利,E 。答(2019 年)。重组蛋白生产的新工具大肠杆菌 :一个5 -年的更新。蛋白质科学28(8):1412- 14 22。
罗森伯格,A 。H . ,拉德,B 。否。,道山,Ç 。,林,S 。w ^ 。,邓恩,J 。Ĵ 。,学生,F 。W. (1987)。通过 T7 RNA 聚合酶选择性表达克隆 DNA 的载体。基因56(1):125 -1 35。
先令,P 。Ĵ 。,米尔扎德,K 。,卡明,A 。Ĵ 。,怀兹海姆,M 。,科克,ž 。和戴利,D 。O. (2020)。pET 表达质粒的改进设计提高了大肠杆菌中的蛋白质产量。公共生物学3(1):214。
希恩,J 。和达尔加诺,L. (1975)。细菌核糖体顺反子特异性的决定因素。自然254(5495):34 -3 8。
席尔瓦-罗查,R 。,马丁内斯,加西亚,ē 。,卡列斯,B 。,查瓦里亚,M 。, Arce-Rodríguez , A . 和德拉斯赫拉斯,A 。,等人。(2013)。标准欧洲载体体系结构 (SEVA):用于分析和部署复杂原核表型的连贯平台。核酸研究41(D1):D666 - D6 75。
沃尔什,G。大规模蛋白质生产。在:蛋白质:生物化学和生物技术,第二版。141 -1 76。
 
版权所有 © 20 21作者;独家被许可人 Bio-protocol LLC。1                                                                                                                             
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shilling, P. J. and Daley, D. O. (2021). Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production. Bio-protocol 11(16): e4133. DOI: 10.21769/BioProtoc.4133.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。