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Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases
精确标记法:通过组合使用II型和IIS型限制性内切核酸酶的新颖无缝蛋白质标记法   

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Aging Cell
May 2016

Abstract

Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, efficiently integrating an array of tags into the gene of interest remains a challenge. Traditional DNA recombinant technology based on type II restriction endonucleases renders protein tagging tedious and inefficient as well as the introduction of an unwanted junction sequence. In our attempt to tag Thrombospondin type 1 domain-containing 1 (THSD1) that we identified as the first intracranial aneurysm gene (Santiago-Sim et al., 2016), we developed a novel precision tagging technique by combinational use of type II and IIS restriction endonucleases (Xu et al., 2017), which generates a seamless clone with high efficiency. Here, we describe a protocol that not only provides a generalized strategy for any gene of interest but also takes its application of 11 different tags in THSD1 as a step-by-step example.

Keywords: DNA cloning (DNA克隆), Protein tagging (蛋白质标记), Type IIS restriction enzyme (IIS型限制酶), Non-palindromic (非回文), THSD1 (THSD1)

Background

Versatile tags with different features serve as a set of tools to dissect protein function molecularly. Various tags such as Green Fluorescence Protein (GFP) tag and its derivatives, tandem affinity purification tags, such as FLAG-HA or ProtA-CBP, have revolutionized the biological research over the years. Some newly developed chemical tags, such as SNAP or CLIP, allow conditional labeling of the protein of interest in a time-controlled fashion (Bodor et al., 2012). However, a method to incorporate as many different tags as possible into a gene of interest efficiently has been poorly developed.

Traditional DNA recombination utilizes type II restriction endonucleases that recognize palindromic sequences. For example, EcoRI recognizes 5’-GAATTC and cleaves inside to make a 3’-AATT sticky end. When a protein needs different tags, it is a tedious and inefficient process that may also lead to the introduction of unwanted junction sequences due to the existence of a restriction recognition sequence. To improve the cloning efficiency, gateway technology takes advantage of another enzyme called integrase, which allows for site-specific recombination (Esposito et al., 2009). However, as patented technology, it requires that many essential reagents be purchased from designated resources. Also, the recognition sequence for integrase imposes a longer unwanted junction sequence between the tag and the protein of interest.

In our recent study to add 11 different tags to the N-terminus of THSD1, a single-span transmembrane protein responsible for cerebral aneurysm pathogenesis (Santiago-Sim et al., 2016), we developed a new cloning strategy by combinational use of type II and type IIS restriction endonucleases (Xu et al., 2017). Unlike type II, type IIS restriction endonucleases recognize non-palindromic sequences and cleave the DNA outside of their recognition site. For example, BsaI recognizes 5’-GGTCTC and cleaves the DNA a nucleotide downstream, resulting in a 5’ overhang 4 nucleotides long, thus making a custom sticky end that matches the gene of interest. Therefore, we can generate a seamless clone by completely eliminating the unwanted junction sequences (Xu et al., 2017). Even more importantly, using type II and type IIS restriction endonucleases in combination makes our method highly compatible with the traditional cloning system that is still widely used by many research labs. In addition, unlike gateway technology, precision tagging does not require designated destination vectors. Since each lab may favor a different set of destination vectors and tags for its gene of interest, our protocol affords researchers great flexibility in making their personalized tagging choices.

Materials and Reagents

  1. PCR tubes (Corning, Axygen®, catalog number: PCR-02-C )
  2. Pipette tips
  3. Razor blade (Fisher Scientific, catalog number: 12-640 )
  4. Microcentrifuge tubes, 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
  5. Petri dish, 100 mm (Fisher Scientific, catalog number: FB0875713 )
  6. DH5α competent cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18265017 )
  7. Plasmids containing different tags:
    pBabe-GFP (Addgene, catalog number: 10668 )
    mCherry-Talin-H-18 (Addgene, catalog number: 62749 )
    Dendra2-Vinculin-N-21 (Addgene, catalog number: 57749 )
    pSNAPf-C1 (Addgene, catalog number: 58186 )
    miniSOG-Zyxin-6 (Addgene, catalog number: 57781 )
  8. Type II restriction endonucleases such as:
    EcoRI-HF (New England Biolabs, catalog number: R3101S )
    BamHI-HF (New England Biolabs, catalog number: R3136S )
    SalI-HF (New England Biolabs, catalog number: R3138S )
    Bsu36I (New England Biolabs, catalog number: R0524S )
  9. Type IIS restriction endonucleases such as:
    BsaI-HF (New England Biolabs, catalog number: R3535S )
    BbsI-HF (New England Biolabs, catalog number: R3539S )
    BsmBI (New England Biolabs, catalog number: R0580S )
  10. LB broth (Fisher Scientific, catalog number: BP1427-500 )
  11. LB agar (Fisher Scientific, catalog number: BP1425-500 )
  12. Ampicillin sodium salt (Fisher Scientific, catalog number: BP1760-5 )
  13. Calf intestinal alkaline phosphatase (CIP) (New England Biolabs, catalog number: M0290S )
  14. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  15. pBS-KSII-4B was modified from pBS-KSII (accession number: X52327) by synonymously destroying BsaI site (Xu et al., 2017)
    Note: It is available upon request and will be deposited to Addgene soon.
  16. pCMV5 (accession number: AF239249, from lab stock and available upon request) and pBabe-puro (Addgene, catalog number: 1764 )
  17. Ultrapure water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  18. Phusion high-fidelity DNA polymerase (New England Biolabs, catalog number: M0530S )
  19. Custom DNA Oligonucleotides (Integrated DNA Technology)
  20. 1x Low-EDTA TE buffer pH 8.0 (Quality Biological, catalog number: 351-324-721 )
  21. 6x Gel loading dye, purple (New England Biolabs, catalog number: B7024S )
  22. Agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17850 )
  23. GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0313 )
  24. 50x TAE (Bio-Rad Laboratories, catalog number: 1610773 )
  25. UltraPure Ethidium Bromide (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15585011 )
  26. QIAquick gel extraction (QIAGEN, catalog number: 28704 )
  27. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
  28. QIAprep spin miniprep kit (QIAGEN, catalog number: 27106 )
  29. Overlapping PCR (Recipe 1)
  30. In vitro digestion by restriction endonucleases (Recipe 2)
  31. Linearization of a vector by restriction endonucleases and dephosphorylation by calf intestinal alkaline phosphatase (CIP) (Recipe 3)
  32. In vitro DNA ligation (Recipe 4)
  33. DNA transformation (Recipe 5)
  34. In vitro DNA oligonucleotide annealing (Recipe 6)
  35. In vitro T4 polynucleotide kinase phosphorylation (Recipe 7)

Equipment

  1. Pipettes
  2. Thermo Cycler (Applied Biosystems) (or any other convention PCR device)
  3. DNA electrophoresis system (Takara Bio, model: Mupid-exU )
  4. 3UV lamp (Fisher Scientific)
  5. Water bath (VWR International), set at 37 °C for enzymatic digestion and 42 °C for DNA transformation into DH5α competent cells
  6. Heat block (VWR International), set at 55 °C for agarose gel dissolution
  7. Air incubator (VWR International), set at 37 °C for bacteria growth on LB plates
  8. Microcentrifuge (Eppendorf, model: 5424 )
  9. Orbital shaker (Forma Scientific), set at 37 °C for bacteria growth in LB medium
  10. ChemiDoc MP imaging system (Bio-Rad Laboratories, model: ChemiDocTM MP )

Software

  1. DNA sequence analysis software: Word, Vector NTI, DNAstar, or other

Procedure

  1. Choosing the appropriate combination of type II and type IIS restriction endonucleases
    1. Choose the gene of interest (GOI). For example, we recently focused on adding different tags to THSD1, a single-span transmembrane protein.
    2. Determine the tags to be inserted. We applied 11 different tags to THSD1 (Table 1).

      Table 1. Information of 11 tags used for THSD1. Information on the amino acids of the tags smaller than 150 bp is shown here, and the addgene number for each tag larger than 150 bp is referenced.


    3. Find type II restriction enzymes that are absent for the GOI and the tags to be inserted. For simplicity, we term them as ‘non-cutters’ hereafter. For human THSD1 (accession number: NM_018676), some non-cutters are available such as BamHI, ClaI, EcoRI, EcoRV, NheI, SalI, XbaI, XhoI, etc. Since the sequences of the tags are custom designed, alternative codon usage can be potentially used to remove the restriction site.
    4. Design the appropriate combination of the identified non-cutters based on pBS-KSII-4B and your destination vector. pBS-KSII-4B is a modified vector for precision tagging (Xu et al., 2017) and it shares the identical multiple cloning site with the widely-used cloning vector pBS-KSII (accession number: X52327). To express tagged THSD1 in mammalian cells, we chose two different destination vectors including pCMV5 and pBabe-puro for transient or stable protein expression, respectively. Accordingly, BamHI, SalI and EcoRI were selected since BamHI/SalI can transfer the insert into pCMV5 while BamHI/EcoRI does the same for pBabe-puro.
    5. Subclone the GOI into pBS-KSII-4B vector using selected non-cutters (Figures 1A to 1B). We termed this new construct as ‘entry clone’ and labeled it as pBS-KSII-4B-GOI. Using regular PCR, we added a BamHI site at the N-terminus, and a sequential SalI-EcoRI site at the C-terminus of THSD1 in pBS-KSII-4B. Therefore, tagged THSD1 in pBS-KSII-4B can work for both pCMV5 and pBabe-puro. If more destination vectors are needed, we may consider readjusting their sequence order of multiple cloning sites to be compatible with pBS-KSII-4B.


      Figure 1. Overview of the workflow for precision tagging by combinational use of type II and type IIS restriction endonucleases. A-E. GOI in different stages is specified in the precision tagging workflow. Type IIS DNA cassette in the GOI is highlighted in the red box. Type IIS restriction endonucleases are used between (C) and (D) and emphasized in blue frame. GOI is the abbreviation of ‘Gene of Interest’.

    6. Determine the position of the tag of interest. In our case, we inserted the tag right after the signal peptide of THSD1 (Figure 2A). In general, tags can be placed before the N-terminal or after the C-terminal of a full-length protein. For a transmembrane protein, tags have to be inserted after the signal peptide if the N-terminal tagging is needed, since they will be cleaved upon entering the lumen of the endoplasmic reticulum. In particular, THSD1 is a type I transmembrane protein, and its N-terminal region will be located outside the cytoplasmic membrane. To avoid any potential interference with THSD1 intracellular signaling transduction, we decided to tag its extracellular domain corresponding to its N-terminal region.
    7. Find a single-cut type II restriction endonuclease that is close to the position of the tag of interest. Henceforth, we will refer to this as a ‘unique cutter’.
    8. Check whether the unique cutter is absent for both the tags and pBS-KSII-4B. For THSD1, we chose Bsu36I that is 455 bp away from the tag position (Figure 2A).


      Figure 2. Schematic of making a template clone from an entry clone of THSD1. A-B. The signal peptide of THSD1 is highlighted in the light blue box while the rest in the orange box containing a green box with the labeled transmembrane domain (TM). BamHI and Bsu36I were selected to replace the original fragment in (A) with the template fragment containing BsaI DNA cassette (highlighted in red box) in (B).

    9. Note that four type IIS restriction endonucleases, including BsaI, BbsI, BsmBI, and BfuAI, are absent for pBS-KSII-4B. All of them cleave DNA outside its non-palindromic recognition sequences, and more product information is available on the New England Biolabs’ website.
    10. Check which type IIS restriction endonuclease is absent for the GOI or the tag. For example, BsaI is absent for THSD1, and different type IIS enzymes may be selected for different tags. In our case, we applied 11 tags (Table 1) and used BsaI, BbsI, and BsmBI, accordingly.
    11. Choose a type IIS DNA cassette. Four type IIS restriction endonuclease-mediated DNA cassettes containing detailed nucleotide information are shown in Figure 3. For THSD1, we chose a BsaI DNA cassette.


      Figure 3. Four kinds of type IIS DNA cassettes for pBS-KSII-4B. (A) BsaI (B) BsmB1 (C) BbsI (D) BfuA1 recognized sequences are indicated in the braces. The cutting positions for them are indicated at both ends of the DNA cassette or the bottom panel.

    12. Record the DNA sequence after inserting the selected type IIS DNA cassette into the tag position of the GOI. Word, Vector NTI, DNAstar, or other DNA sequence analysis software is helpful. We named the sequence that contains the type IIS DNA cassette from one end to the site of unique cutter in the GOI as ‘template fragment’.

  2. Construction of a template clone by type II restriction endonucleases
    1. Perform overlapping PCR to make a template fragment. For THSD1, we performed the following experiment as indicated in Figure 4 and Recipe 1 using Thermo Cycler.
      1. Order two oligos (BamHI-THSD1-F and Bsu36I-THSD1-R) that match both ends of the template fragment, with additional type II restriction enzymes such as BamHI or Bsu36I.
      2. Two internal overlapping oligos contain 18-24 nt of the left (light blue box) and right side (orange box) of the type IIS DNA cassette (red box), which is in total around 54-68 nt (BsaI-OL-F and BsaI-OL-R). The sequence information for above oligos is listed in Table 2.


        Figure 4. Schematic of making a template fragment of THSD1 from overlapping PCR. Detailed information for the oligos is provided in Table 2. BamHI was added to the forward primer for NT-fragment PCR. Overlapping PCR contains part of a signal peptide (highlighted in the light blue box), BsaI DNA cassette (selected from Figure 2, highlighted in the red box), and part of THSD1 coding sequence (highlighted in the orange box). Bsu36I is in the middle of the reserve primer for CT fragment PCR. The detailed information of the final PCR product is indicated in the region of the template clone between two black lines.

        Table 2. Information of overlapping PCR. BsaI DNA cassette is highlighted in red letters. BamHI and Bsu36I cutting sites are italicized. Nucleotides are colored according to Figure. OL is short for ‘over-lapping’.


    2. Run the PCR product on a gel using the Mupid-exU DNA electrophoresis system, and check the size of amplified NT and CT fragment followed by gel purification (QIAquick gel extraction kit). Purify the DNA band of interest using QIAquick gel extraction kit (melt the gel in a 55 °C heat block). Next, perform overlapping PCR by mixing NT and CT fragment as indicated in Recipe 1 and gel purify the template fragment of correct size in the same way.
    3. Alternatively, order the template fragment from Integrated DNA Technology (IDT) or other companies that provide similar services involving large DNA fragment synthesis. We ordered it with additional BamHI at the N-terminus, Bsu36I at the C-terminus, and BsaI DNA cassette right after the signal peptide of THSD1. Use the type II restriction endonucleases to digest the template fragment obtained either from overlapping PCR or the commercial gene synthesis. For THSD1, digest both fragments with BamHI and Bsu36I for 2 h at 37 °C using a water bath (Recipe 2 for enzymatic digestion).
    4. Linearize pBS-KSII-4B-GOI with the same restriction endonucleases that are used for the template fragment, followed by calf intestinal phosphatase treatment (Recipe 3 for vector linearization). For example, we linearized pBS-KSII-4B-THSD1 with BamHI and Bsu36I.
    5. Ligate the digested template fragment with linearized pBS-KSII-4B-GOI (Recipe 4 for DNA ligation). Accordingly, we joined the template fragment with the above linearized pBS-KSII-4B-THSD1 (Figure 2).
    6. Transform DH5α competent cells with the ligation product and spread them on LB agar plates containing 100 µg/ml ampicillin (Recipe 5 for DNA transformation).
    7. Incubate the plates at 37 °C air incubator overnight.
    8. Pick up several colonies on each plate and inoculate them separately into a clean tube containing 2-5 ml LB medium with 100 µg/ml ampicillin.
    9. Grow the bacteria in an orbital shaker with a shaking speed of 220 rpm at 37 °C overnight.
    10. Extract the plasmids from the overnight cultures using QIAprep spin miniprep kit.
    11. Confirm the correct clones by restriction analysis and Sanger sequencing.

  3. Construction of a tag clone by type IIS restriction endonucleases
    1. Define the tag sequence using DNA analysis software, such as Word or DNAstar.
    2. For the tags that are smaller than 150 bp, such as HA, Myc or FLAG, order two annealing oligos as indicated in Figure 5A. So far, a 200 bp long oligo is the maximum length that can be provided commercially, and that is why 150 bp was chosen as an arbitrary cutoff. It still leaves extra spaces for adding gene-specific DNA sequences at both ends.
    3. For the tags that are larger than 150 bp, such as GFP, miniSOG, or SNAPf, order two amplifying oligos with type IIS restriction endonuclease sequences at both ends as indicated in Figure 5B. To amplify these tags, perform PCRs under the same condition for each tag by following a standard protocol:
      Template DNA
      1 ng
      Amplifying Oligo-F (50 µM)
      1 µl
      Amplifying Oligo-R (50 µM)
      1 µl
      10x HF buffer
       5 µl
      dNTP (25 µM)
      1 µl
      Phusion high-fidelity DNA polymerase
      0.5 µl
      H2O added to total
       50 µl
      Initial denaturation: 98 °C 30 sec; 28 cycles: 98 °C 10 sec, 60 °C 15 sec, 72 °C 1 min; final extension: 72 °C 5 min.


      Figure 5. Generation of the tag fragment with custom sticky ends. A. The sense annealing oligo (annealing oligo-S) is composed of 5’-AGCT (letters in red) and tag-specific sequence (letters in blue); the anti-sense annealing oligo (annealing oligo-AS) is composed of 5’-ATTC (letters in green) and tag-specific sequence (letters in blue), as shown in the right table. B. Both amplifying oligos contain a BsaI recognition site (letters in black), THSD1-specific sticky end (letters in red or green), and tag-specific sequences (letters in blue), as detailed on the right table.

    4. Following Step C2, oligos are annealed and phosphorylated by T4 polynucleotide kinase.
    5. Following Step C3, amplified PCR products are digested by type IIS enzymes for 2 h at 37 °C (Recipe 2).
    6. Linearize the template clone of pBS-KSII-4B-GOI containing type IIS DNA cassette by the predesigned type IIS restriction endonucleases. For example, pBS-KSII-4B-THSD1 containing BsaI cassette is digested by BsaI for 2 h at 37 °C, and dephosphorylated by calf intestinal alkaline phosphatase (Recipe 2 and Figures 6A-6B).
    7. Ligate the tag fragments with two sticky ends that are generated at Step C4 or Step C5, into the linearized template clone from Step C6 (Figures 6B-6C).


      Figure 6. Schematic of making a tag clone from a template clone of THSD1. A-B. The template clone contains two THSD1-specific sticky ends (letters in red or green). C. All above tags containing a THSD1-specific sticky end and 5’-phosphorylation are joined with the linearized template clone by T4 DNA ligase, thus generating a seamless tag clone.

    8. Transform DH5α competent cells with the ligation product and spread them on LB agar plates containing 100 µg/ml ampicillin (Recipe 5 for DNA transformation).
    9. Grow the plates at 37 °C air incubator overnight.
    10. Pick up several colonies on each plate and inoculate them separately into a clean tube containing 2-5 ml LB medium with 100 µg/ml ampicillin.
    11. Grow the bacteria in an orbital shaker with a shaking speed of 220 rpm at 37 °C overnight.
    12. Extract the plasmids from the overnight cultures using QIAprep spin miniprep kit.
    13. Confirm the correct clones by restriction analysis and Sanger sequencing.

  4. Construction of a destination clone by type II restriction endonucleases
    1. Linearize your destination vector with the preselected type II restriction enzymes from Procedure A. For instance, we linearized pCMV5 with BamHI and SalI, or pBabe-puro with BamHI and EcoRI (Recipe 3).
    2. Cut your tagged GOI from previous tag clones in Procedure C with the same enzymes, followed by running agarose gel electrophoresis and extracting DNA band of correct size from the gel. For pBS-KSII-4B-THSD1 with different tags, we cut tagged THSD1 by either BamH/SalI or BamHI/EcoRI, and recovered the DNA band of correct size by gel purification (QIAquick gel extraction kit).
    3. Ligate the tagged GOI with the linearized destination vector by compatible sticky ends. In Figure 6, how tagged THSD1 are transferred from the tag clone into the destination clone like pCMV5 are shown (Figure 7).


      Figure 7. Schematic of making a destination clone from a tag clone of THSD1. BamHI and SalI were selected to transfer the tagged THSD1 in pBS-KSII-4B to pCMV5 vector using traditional cloning method.

    4. Transform DH5α competent cells with the ligation product and spread them on LB agar plates containing 100 µg/ml ampicillin (Recipe 5 for DNA transformation).
    5. Incubate the plates at 37 °C air incubator overnight.
    6. Pick up several colonies on each plate and inoculate them separately into a clean tube containing 2-5 ml LB medium with 100 µg/ml ampicillin.
    7. Grow the bacteria in an orbital shaker with a shaking speed of 220 rpm at 37 °C overnight.
    8. Extract the plasmids from the overnight cultures using QIAprep spin miniprep kit.
    9. Confirm the correct clones by restriction analysis and Sanger sequencing.

Data analysis

All clones at different stages (Figure 1) can be characterized by restriction endonucleases and followed by agarose gel electrophoresis. Gel images are recorded in the ChemiDoc imaging system. The shuttling vector pBS-KSII-4B does not contain BsaI and other three type IIS enzymes, such as BbsI, BsmBI, and BfuA1, are naturally absent (Figure 8).


Figure 8. Features of pBS-KSII-4B. BsaI-recognized ‘GGTCTC’ was mutated to ‘GGTCAC’ (highlighted by blue underline), which synonymously destroyed BsaI site.

  1. For further verification of different clones, DNA needs to be sent out for local sequencing service. Softwares such as Vector NTI or DNAstar is helpful for DNA sequence analyses.
  2. Tagged GOI in destination vectors can be functionally evaluated. For example, THSD1 with different tags can be confirmed by immunoblotting against overexpressed HEK293T cells.

Notes

  1. Unsuccessful PCR amplification of template
    Unsuccessful PCR includes two scenarios: lack of amplified fragment of correct size, or weak detection of target band in addition to multiple non-specific fragments. For the former situation, make sure that PCR reaction system is still working such as the expiration date of the enzyme, buffer or dNTP, which can be assessed by using other primers and template that has been successfully amplified previously. Double check the new primers to see if they match the template in the right orientation. On occasions, repetitive sequences in the primers that form special secondary structures will interfere with effective PCR amplification, which can be improved by adding 5 M betaine into the reaction system (final concentration of betaine is 3 M). For the latter issue such as detection of weak amplification of target band co-existing with other non-specific fragments, make sure that the Tm values of each pair of primers are close. If not, adjust them simply by changing the length of primers. Besides, increasing the annealing temperature will help increase PCR specificity in general.
  2. Only empty vector produced by ligation
    It may be caused by incomplete digestion of a DNA fragment of interest. Reducing the amount of DNA fragment, increasing the units of digestive enzymes, or maximizing their activities using a compatible buffer may help. New England Biolabs has produced many new versions of the enzymes that cut the same DNA sequence and have 100% activity in different buffers in comparison with the old versions. Therefore, these new enzymes also called high-fidelity enzymes provide more options on buffer selections when maximized activities of double enzymatic digestion are needed.

Recipes

  1. Overlapping PCR
    1. The below reaction is for N-terminal (NT) fragment PCR. Template DNA 1 ng
      Oligo-F (50 µM)
      1 µl
      Overlapping-R (50 µM)
      1 µl
      10x HF buffer
      5 µl
      dNTP (25 µM)
      1 µl
      Phusion high-fidelity DNA polymerase
      0.5 µl
      H2
      added to total 50 µl
      Note: The annealing temperature is 55-65 °C with 28 cycles.
    2. For C-terminal (CT) fragment PCR, use oligo-R and overlapping-F primers
    3. Gel purify the amplified NT and CT fragment using QIAquick gel extraction kit
    4. Mix purified fragments in a ratio of 1:1 as a new template DNA and run overlapping PCR as follows:
      Mixed template DNA
      around 100 ng
      10x HF buffer
      5 µl
      dNTP (25 µM)
      1 µl
      Phusion high-fidelity DNA polymerase
      0.5 µl
      H2O
      added to total 50 µl
    5. Run 10 cycles with annealing temperature being 55 °C or higher. Continue to run extra 20 cycles after adding oligo-F (1 µl) and oligo-R (1 µl) to the reaction system
    6. Extract the fragment of correct size with gel purification
  2. In vitro digestion by restriction endonucleases
    DNA
    1 µg
    10x buffer
    5 µl
    Restriction endonuclease #1
    0.75 µl
    Restriction endonuclease #2
    0.75 µl
    H2O
    added to total 50 µl
    Incubate at 37 °C for 2 h
    Note: Optimal temperature for digestion may vary for different enzymes such as 55 °C for BsmBI.
  3. Linearization of a vector by restriction endonucleases and dephosphorylation by calf intestinal alkaline phosphatase (CIP)
    1. Linearization of a vector: same as Recipe 2
    2. Vector de-phosphorylation: after a vector linearization, 1 µl CIP was added and incubated at 37 °C for 40 min
  4. In vitro DNA ligation
    DNA insert
    12.5 µl
    Linearized vector
    1 µl
    10x T4 DNA ligase buffer
    1.5 µl
    Room temperature, 1 h
    Note: Negative control is the ligation of linearized vector alone by replacing DNA insert with water.
  5. DNA transformation
    1. Ligation product is incubated with DH5α competent cells on ice for 30 min
    2. Heat shock at 42 °C for 1 min
    3. Cells are allowed to recover for 1 h at 37 °C with a shaking speed of 220 rpm prior to plating on LB agar with appropriate antibiotics
  6. In vitro DNA oligonucleotide annealing
    1. Each oligo was dissolved in 1x low EDTA TE buffer to 50 µM
    2. Mix equal volume of annealing oligos, and put them at 95 °C heat block for 5 min, and turn it off to cool slowly to room temperature
  7. In vitro T4 polynucleotide kinase phosphorylation
    Annealed oligos
    1 µl
    10x T4 DNA ligase buffer
    1 µl
    T4 polynucleotide kinase
    1 µl
    H2O
    added to total 10 µl
    Incubate at 37 °C for 1 h
    Store at -20 °C
    Note: For later DNA ligation, the phosphorylated oligos need to be diluted 1:500 before use.

Acknowledgments

We appreciate the critical proofreading of this manuscript by Dr. Joanna O’Leary. The project was supported by RO3NS087416 from the US National Institute of Health.
Conflict of interest statement: The authors declare no conflict of interest or competing interests.

References

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  6. Xu, X., Song, Y., Li, Y., Chang, J., Zhang, H. and An, L. (2010). The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification. Protein Expr Purif 72(2): 149-156.

简介

由于各种标签的存在,蛋白质标签是一种对感兴趣的蛋白质的生物学功能进行全面分析的有力工具。在某些情况下,例如跟踪活细胞中的蛋白质动态变化或由于缺乏最佳抗体而添加肽表位,这变得不可或缺。然而,将一系列标签有效地整合到感兴趣的基因中仍然是一个挑战。基于II型限制性内切核酸酶的传统DNA重组技术使得蛋白质标记繁琐且效率低下以及引入不需要的连接序列。我们试图标记我们确定为第一个颅内动脉瘤基因的血小板反应蛋白1型结构域1(THSD1)(Santiago-Sim等人,2016),我们开发了一种新型精确标记技术,组合使用II型和IIS限制性核酸内切酶(Xu等人,2017),其产生高效率的无缝克隆。在这里,我们描述了一个协议,不仅为任何感兴趣的基因提供了一个广义的策略,而且还将THSD1中的11个不同标签的应用作为一个循序渐进的例子。


【背景】具有不同特征的多功能标签可以作为一组分析蛋白质功能的工具。诸如绿色荧光蛋白(GFP)标签及其衍生物,串联亲和纯化标签(如FLAG-HA或ProtA-CBP)的各种标签多年来革新了生物学研究。一些新开发的化学标签,如SNAP或CLIP,允许以时间控制的方式有条件地标记感兴趣的蛋白质(Bodor等人,2012)。然而,有效地将尽可能多的不同标签整合到感兴趣的基因中的方法发展不足。

传统的DNA重组利用识别回文序列的II型限制性内切核酸酶。例如,Eco RI识别5'-GAATTC并切割内部以产生3'-AATT粘性末端。当蛋白质需要不同的标签时,由于存在限制识别序列,这是一个繁琐而低效的过程,也可能导致不需要的连接序列的引入。为了提高克隆效率,门户技术利用了另一种称为整合酶的酶,其允许进行位点特异性重组(Esposito等人,2009)。但是,作为专利技术,需要从指定资源购买许多必要的试剂。而且,整合酶的识别序列在标签和感兴趣的蛋白质之间施加更长的不需要的连接序列。

在我们最近的研究中,在负责脑动脉瘤发病机制的单跨膜蛋白质THSD1(Santiago-Sim等人,2016)的N端添加了11个不同的标签,我们开发了一种新的通过组合使用II型和IIS型限制性核酸内切酶克隆策略(Xu等人,2017)。与II型不同,IIS型限制性核酸内切酶识别非回文序列并在识别位点之外切割DNA。例如,Bsa识别5'-GGTCTC并将DNA下游的一个核苷酸切割,导致5'突出长度为4个核苷酸长,从而形成与感兴趣的基因匹配的定制粘端。因此,我们可以通过完全消除不需要的结点序列来产生无缝的克隆(Xu等人,2017)。更重要的是,将II型和IIS型限制性内切酶结合使用,使得我们的方法与许多研究实验室仍然广泛使用的传统克隆系统高度兼容。另外,与网关技术不同,精确标记不需要指定的目标向量。由于每个实验室都可能为其感兴趣的基因偏爱一组不同的目标载体和标签,因此我们的协议为研究人员提供了极大的灵活性,使其可以选择个性化的标签。

关键字:DNA克隆, 蛋白质标记, IIS型限制酶, 非回文, THSD1

材料和试剂

  1. PCR管(Corning,Axygen,目录号:PCR-02-C)
  2. 移液器提示
  3. 剃刀刀片(Fisher Scientific,目录号:12-640)
  4. 微量离心管,1.5ml(Fisher Scientific,目录号:05-408-129)
  5. 培养皿,100毫米(Fisher Scientific,目录号:FB0875713)
  6. DH5α感受态细胞(Thermo Fisher Scientific,Invitrogen TM,产品目录号:18265017)
  7. 包含不同标签的质粒:
    pBabe-GFP(Addgene,目录号:10668)
    mCherry-Talin-H-18(Addgene,目录号:62749)
    Dendra2-Vinculin-N-21(Addgene,目录号:57749)
    pSNAPf-C1(Addgene,目录号:58186)
    miniSOG-Zyxin-6(Addgene,目录号:57781)
  8. II型限制性核酸内切酶如:
    Eco-RI-HF(New England Biolabs,目录号:R3101S)
    Bam HI-HF(New England Biolabs,目录号:R3136S)
    Sal-I-HF(New England Biolabs,目录号:R3138S)
    Bsu 36I(New England Biolabs,目录号:R0524S)
  9. IIS型限制性内切酶如:
    Bsa I-HF(New England Biolabs,目录号:R3535S)
    Bbs I-HF(New England Biolabs,目录号:R3539S)
    Bsm(新英格兰生物实验室,目录号:R0580S)
  10. LB肉汤(Fisher Scientific,目录号:BP1427-500)
  11. LB琼脂(Fisher Scientific,目录号:BP1425-500)
  12. 氨苄青霉素钠盐(Fisher Scientific,目录号:BP1760-5)
  13. 小牛肠碱性磷酸酶(CIP)(新英格兰生物实验室,目录号:M0290S)
  14. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  15. 通过同义地破坏BsaI位点(Xu等人,2017),从pBS-KSII(登记号:X52327)修改pBS-KSII-4B。 注:它可以根据要求提供,并很快将存入Addgene。
  16. pCMV5(登录号:AF239249,来自实验室原液,可根据要求提供)和pBabe-puro(Addgene,目录号:1764)
  17. 超纯水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  18. Phusion高保真DNA聚合酶(New England Biolabs,目录号:M0530S)
  19. 定制DNA寡核苷酸(集成DNA技术)
  20. 1x低-EDTA TE缓冲液pH8.0(Quality Biological,目录号:351-324-721)
  21. 6X凝胶上样染料,紫色(新英格兰生物实验室,目录号:B7024S)
  22. 琼脂糖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17850)
  23. GeneRuler 1kb DNA ladder(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SM0313)
  24. 50倍TAE(Bio-Rad Laboratories,目录号:1610773)
  25. UltraPure Ethidium Bromide(Thermo Fisher Scientific,Invitrogen TM,目录号:15585011)
  26. QIAquick凝胶提取(QIAGEN,目录号:28704)
  27. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  28. QIAprep spin miniprep kit(QIAGEN,目录号:27106)
  29. 重叠PCR(配方1)
  30. 限制性内切酶体外消化(方案2)
  31. 通过限制性内切核酸酶线性化载体并通过小牛肠碱性磷酸酶(CIP)去磷酸化(方案3)
  32. 体外DNA结扎(方案4)
  33. DNA转化(方案5)
  34. 体外DNA寡核苷酸退火(方案6)
  35. 体外T4多核苷酸激酶磷酸化(方案7)

设备

  1. 移液器
  2. Thermo Cycler(应用生物系统公司)(或任何其他常规PCR设备)
  3. DNA电泳系统(Takara Bio,型号:Mupid-exU)
  4. 3UV灯(Fisher Scientific)
  5. 水浴(VWR国际),设置在37°C的酶消化和42°C的DNA转化DH5α感受态细胞
  6. 加热块(VWR国际),设定在55°C琼脂糖凝胶溶解
  7. 空气培养箱(VWR国际),设置在37°C的LB平板上的细菌生长
  8. 微量离心机(Eppendorf,型号:5424)
  9. 轨道振荡器(Forma Scientific),设置在37℃,用于LB培养基中的细菌生长
  10. ChemiDoc MP成像系统(Bio-Rad Laboratories,型号:ChemiDoc TM TM)

软件

  1. DNA序列分析软件:Word,Vector NTI,DNAstar或其他

程序

  1. 选择II型和IIS型限制性核酸内切酶的适当组合
    1. 选择感兴趣的基因(GOI)。例如,我们最近专注于为THSD1(一种跨膜跨膜蛋白)添加不同的标签。
    2. 确定要插入的标签。我们对THSD1应用了11个不同的标签(表1)。

      表1.用于THSD1的11个标签的信息这里显示了小于150bp的标签的氨基酸的信息,并且参考了大于150bp的每个标签的基因编号。


    3. 找到对于GOI而言不存在的II型限制性酶以及待插入的标签。为了简单起见,我们在下文中将它们称为“非切割器”。对于人THSD1(登录号:NM_018676),可以使用一些非切割器,例如Bam HI,Cla C I,Eco RI,EM > Eco RV, Nhe I, Sal I, Xba I, Xho >等。由于标签的序列是定制设计的,因此可以使用替代密码子使用来去除限制性位点。
    4. 根据pBS-KSII-4B和您的目的地载体设计已鉴定的非切割器的适当组合。 pBS-KSII-4B是用于精确标记的修饰载体(Xu等人,2017),它与广泛使用的克隆载体pBS-KSII(登录号:X52327)共享相同的多克隆位点)。为了在哺乳动物细胞中表达标记的THSD1,我们选择了两种不同的目标载体,包括pCMV5和pBabe-puro分别用于瞬时或稳定的蛋白质表达。因此,选择Bam HI,Sal I和Eco EcoRI,因为Bam HI / Sal 我可以将插入片段转移到pCMV5中,而BamHI / EcoRI对于pBabe-puro片段也是一样的。
    5. 使用选择的非切割器将GOI亚克隆到pBS-KSII-4B载体中(图1A至1B)。我们称这个新的构建为“入门克隆”,并将其标记为pBS-KSII-4B-GOI。使用常规PCR,我们在N-末端添加了一个BamHI位点,并在C-末端添加了一个连续的Sal-I-Eco EcoRI位点,在pBS-KSII-4B中的THSD1的末端。因此,在pBS-KSII-4B中标记的THSD1可以用于pCMV5和pBabe-puro。如果需要更多的目标载体,我们可能会考虑重新调整它们与多个克隆位点的序列顺序,以便与pBS-KSII-4B兼容。


      图1.通过组合使用II型和IIS型限制性核酸内切酶进行精确标记的工作流程概述。 A-E。在精确标记工作流程中指定了不同阶段的GOI。红色框中突出显示了GOI中的IIS DNA盒。 (C)和(D)之间使用IIS型限制性核酸内切酶,并在蓝框中强调。 GOI是“感兴趣的基因”的缩写。

    6. 确定感兴趣的标签的位置。在我们的例子中,我们在THSD1的信号肽之后插入了标签(图2A)。通常,标签可以放置在全长蛋白质的N端之前或C端之后。对于跨膜蛋白,如果需要N-末端标签,则必须在信号肽之后插入标签,因为它们在进入内质网腔时将被切割。具体而言,THSD1是I型跨膜蛋白,其N端区域位于细胞质膜外。为了避免任何潜在的干扰THSD1细胞内信号转导,我们决定标记其对应于其N端区域的细胞外结构域。
    7. 找到接近标签位置的单切II型限制性内切酶。今后,我们将把这称为“独特的刀具”。
    8. 检查标签和pBS-KSII-4B是否缺少独特的切刀。对于THSD1,我们选择了距离标签位置455bp的Bsu36I(图2A)。


      图2.从THSD1的入门克隆中制作模板克隆的示意图 A-B。 THSD1的信号肽在浅蓝色框中突出显示,而其余的在橙色框中含有带有标记的跨膜结构域(TM)的绿色框。 (A)中的Bam HI和Bsu 36I被替换为含有BsaI DNA盒的模板片段(以红色突出显示(B)中的框)。

    9. 注意,四种IIS限制性核酸内切酶,包括Bsa I,Bbs I,Bsm II和Bfu AI,缺少pBS-KSII-4B。它们都在非回文识别序列之外切割DNA,更多的产品信息可以在New England Biolabs的网站上找到。
    10. 检查哪种类型的IIS限制性内切核酸酶不存在于GOI或标签中。例如,对于THSD1,缺少Bsa I,对于不同的标签可以选择不同类型的IIS酶。在我们的例子中,我们应用了11个标签(表1),并相应地使用了 Bsa I和 Bsm BI。
    11. 选择一个IIS型DNA盒。图3显示了四种含有详细核苷酸信息的IIS型限制性核酸内切酶介导的DNA盒。对于THSD1,我们选择了一种BsaI DNA盒。


      (A)Bsa B(B)Bsm B1(C) )Bbs I(D) A1在花括号中标出了A1识别的序列。
      在DNA盒或底部面板的两端均标注切割位置
    12. 将选择的IIS DNA盒插入GOI的标签位置后记录DNA序列。 Word,Vector NTI,DNAstar或其他DNA序列分析软件是有帮助的。我们将包含IIS型DNA序列的序列命名为“模板片段”,从一端到GOI独特切片的位置。

  2. 通过II型限制性核酸内切酶构建模板克隆
    1. 执行重叠PCR来制作模板片段。对于THSD1,我们使用Thermo Cycler进行了如图4和Recipe 1所示的以下实验。
      1. 与模板片段两端匹配的两个寡核苷酸(Bam HI-THSD1-F和Bsu 36I-THSD1-R)与另外的II型限制性内切酶如 Bam HI或 Bsu 36I。
      2. 两个内部重叠的寡核苷酸包含IIS DNA盒(红色框)的左侧(浅蓝色框)和右侧(橙色框)的18-24nt,总共大约54-68nt( / I-OL-F和 Bsa I-OL-R)。表2列出了上述寡核苷酸的序列信息。


        图4.由重叠PCR制备THSD1的模板片段的示意图表2中提供了寡聚物的详细信息。将BamHI添加到NT的正向引物片段PCR。重叠PCR包含部分信号肽(在浅蓝色框中突出显示),BsaI DNA盒(选自图2,突出显示在红色框中)和部分THSD1编码序列(突出显示在橙色框).Bsu 36I位于CT片段PCR的保留引物的中间。
        最后PCR产物的详细信息在两条黑线之间的模板克隆的区域中显示
        表2.重叠PCR的信息。 Bsa I DNA盒以红色字母突出显示。 Bam HI和 Bsu 36I切割位置用斜体表示。核苷酸根据图被着色。 OL是“重叠”的缩写。


    2. 使用Mupid-exU DNA电泳系统在凝胶上运行PCR产物,并检查扩增的NT和CT片段的大小,然后进行凝胶纯化(QIAquick凝胶提取试剂盒)。使用QIAquick凝胶提取试剂盒纯化目的DNA条带(在55°C加热块中融化凝胶)。接下来,通过混合NT和CT片段进行重叠PCR(如配方1所示),并以相同的方式凝胶纯化正确大小的模板片段。
    3. 或者,从Integrated DNA Technology(IDT)或其他提供大片段DNA合成相似服务的公司购买模板片段。我们在N末端添加了额外的Bam HI,在C末端添加了Bsu 36I,以及在B末端添加了Bsa I DNA盒THSD1的信号肽。使用II型限制性内切酶消化由重叠PCR或商业基因合成获得的模板片段。对于THSD1,使用水浴(方案2用于酶消化),在37℃下用BamHI和Bsu 36I消化两个片段2小时。
    4. 使用用于模板片段的相同限制性内切核酸酶使pBS-KSII-4B-GOI线性化,接着小牛肠磷酸酶处理(用于载体线性化的配方3)。例如,我们使用Bam HI和Bsu 36I线性化pBS-KSII-4B-THSD1。
    5. 用线性化的pBS-KSII-4B-GOI(用于DNA连接的配方4)将消化的模板片段连接起来。因此,我们加入了上述线性化的pBS-KSII-4B-THSD1模板片段(图2)。
    6. 用连接产物转化DH5α感受态细胞并将其铺在含有100μg/ ml氨苄青霉素的LB琼脂平板上(DNA转化方案5)。
    7. 37°C空气培养箱孵育过夜。
    8. 挑取每个板上的几个殖民地,并分别接种到一个干净的管,含有2-5毫升含100微克/毫升氨苄青霉素的LB培养基。

    9. 在37℃振荡速度为220rpm的定轨振荡器中培养细菌过夜

    10. 使用QIAprep spin miniprep试剂盒从过夜培养物中提取质粒

    11. 通过限制分析和Sanger测序确认正确的克隆
  3. IIS型限制性核酸内切酶构建标签克隆

    1. 使用DNA分析软件(如Word或DNAstar)定义标签序列
    2. 对于小于150bp的标签,如HA,Myc或FLAG,可订购两个退火寡核苷酸,如图5A所示。到目前为止,200bp长的寡核苷酸是商业上可以提供的最大长度,这就是为什么选择150bp作为任意截断值的原因。它仍然留下额外的空间用于在两端添加基因特异性DNA序列。
    3. 对于大于150bp的标签,例如GFP,miniSOG或SNAPf,在两端订购两个具有IIS型限制性核酸内切酶序列的扩增寡核苷酸,如图5B所示。要扩大这些标签,按照标准协议在相同条件下对每个标签进行PCR:
      模板DNA
      1纳克
      放大Oligo-F(50μM)
      1微升
      放大Oligo-R(50μM)
      1微升
      10倍的HF缓冲液
        5微升
      dNTP(25μM)
      1微升
      Phusion高保真DNA聚合酶
      0.5微升
      H 2 添加到总共
        50微升
      初始变性:98℃30秒; 28个循环:98℃10秒,60℃15秒,72℃1分钟;最终延伸:72°C 5分钟。


      图5.具有定制粘端的标签片段的生成A.正义退火寡核苷酸(退火寡聚-S)由5'-AGCT(红色字母)和标签特异性序列(蓝色字母);如右表所示,反义退火寡核苷酸(退火寡聚-AS)由5'-ATTC(绿色字母)和标签特异性序列(蓝色字母)组成。 B.两种扩增寡核苷酸均含有BsaI识别位点(黑色字母),THSD1特异性粘性末端(红色或绿色字母)和标签特异性序列(蓝色字母),如详述在右边的桌子上。

    4. 在步骤C2之后,使寡核苷酸退火并通过T4多核苷酸激酶磷酸化。
    5. 在步骤C3之后,扩增的PCR产物通过IIS型酶在37℃消化2小时(方案2)。
    6. 通过预先设计的IIS型限制性核酸内切酶线性化含有IIS型DNA盒的pBS-KSII-4B-GOI的模板克隆。例如,含有BsaI I盒的pBS-KSII-4B-THSD1在37℃下用BsaI消化2小时,并用小牛肠碱性磷酸酶去除磷酸化(配方2和图6A-6B)。
    7. 将具有在步骤C4或步骤C5生成的两个粘性末端的标签片段导入来自步骤C6的线性化模板克隆(图6B-6C)。


      图6.从THSD1的模板克隆中制作标签克隆的示意图。 A-B。模板克隆包含两个THSD1特定的粘性末端(红色或绿色字母)。 C.所有含有THSD1特异性粘端和5'-磷酸化的标签通过T4DNA连接酶与线性化模板克隆连接,从而产生无缝标签克隆。

    8. 用连接产物转化DH5α感受态细胞并将其铺在含有100μg/ ml氨苄青霉素的LB琼脂平板上(DNA转化方案5)。
    9. 在37℃空气培养箱中培养平板过夜。
    10. 挑取每个板上的几个殖民地,并分别接种到一个干净的管,含有2-5毫升含100微克/毫升氨苄青霉素的LB培养基。

    11. 在37℃振荡速度为220rpm的定轨振荡器中培养细菌过夜

    12. 使用QIAprep spin miniprep试剂盒从过夜培养物中提取质粒
    13. 通过限制性分析和Sanger测序确认正确的克隆。

  4. 通过II型限制性核酸内切酶构建目标克隆
    1. 将目的载体线性化到预先选择的来自程序A的II型限制性内切酶。例如,我们使用BamHI和SalI或pBabe-puro将pCMV5线性化, Bam HI和 Eco RI(配方3)。
    2. 使用相同的酶从过程C中的以前的标签克隆中切下标签的GOI,接着进行琼脂糖凝胶电泳并从凝胶中提取正确大小的DNA带。对于带有不同标签的pBS-KSII-4B-THSD1,我们通过 Bam H / Sal I或 Bam HI / Eco RI,通过凝胶纯化(QIAquick凝胶提取试剂盒)回收正确大小的DNA条带。
    3. 通过兼容的粘性末端使用线性目标向量来标记已标记的GOI。在图6中,显示了如何将标记的THSD1从标记克隆转移到如pCMV5的目标克隆中(图7)。


      图7.从THSD1的标记克隆中生成目的地克隆的示意图。使用传统的克隆方法,选择Bam HI和Sal I以将pBS-KSII-4B中的标记的THSD1转移到pCMV5载体中。

    4. 用连接产物转化DH5α感受态细胞并将其铺在含有100μg/ ml氨苄青霉素的LB琼脂平板上(DNA转化方案5)。
    5. 37°C空气培养箱孵育过夜。
    6. 挑取每个板上的几个殖民地,并分别接种到一个干净的管,含有2-5毫升含100微克/毫升氨苄青霉素的LB培养基。

    7. 在37℃振荡速度为220rpm的定轨振荡器中培养细菌过夜
    8. 使用QIAprep spin miniprep试剂盒从过夜培养物中提取质粒。

    9. 通过限制分析和Sanger测序确认正确的克隆

数据分析

所有处于不同阶段的克隆(图1)均可通过限制性内切核酸酶进行表征,然后进行琼脂糖凝胶电泳。凝胶图像记录在ChemiDoc成像系统中。穿梭载体pBS-KSII-4B不含有BsaI和其他三种IIS酶,如BsmI,BsmBI和 Bfu A1,自然不存在(图8)。


图8. pBS-KSII-4B的特征 Bsa I-识别的“GGTCTC”突变为“GGTCAC”(由蓝色下划线突出显示),其同义地破坏 > Bsa 我的网站。

  1. 为了进一步验证不同的克隆,需要发送DNA用于本地测序服务。 Vector NTI或DNAstar等软件有助于DNA序列分析。
  2. 在目标载体中标记的GOI可以进行功能评估。例如,具有不同标签的THSD1可以通过针对过度表达的HEK293T细胞进行免疫印迹来确认。

笔记

  1. 不成功的PCR扩增模板
    不成功的PCR包括两种情况:缺少正确大小的扩增片段,或者除了多个非特异性片段之外,还没有检测到目标条带。对于前一种情况,确保PCR反应体系仍在工作,如酶的使用日期,缓冲液或dNTP,这可以通过使用其他已经成功扩增的引物和模板来评估。仔细检查新的引物,看它们是否与正确方向的模板匹配。有时,形成特殊二级结构的引物中的重复序列将干扰有效的PCR扩增,通过向反应体系中加入5M甜菜碱(甜菜碱的最终浓度为3M)可以改善这种反应。对于诸如检测与其他非特异性片段共存的目标条带弱扩增的后一问题,确保每对引物的Tm值接近。如果不是,只需通过改变引物长度来调整它们。此外,提高退火温度将有助于提高PCR特异性。
  2. 只有结扎产生的空载体
    这可能是由于感兴趣的DNA片段的不完全消化引起的。减少DNA片段的数量,增加消化酶的单位或使用相容的缓冲液使其活性最大化可能有帮助。新英格兰生物实验室已经产生了许多新版本的酶,与旧版本相比,它们切割相同的DNA序列并且在不同的缓冲液中具有100%的活性。因此,当需要双酶消化的最大化活性时,这些也称为高保真酶的新酶在缓冲液选择上提供了更多的选择。

食谱

  1. 重叠PCR
    1. 下面的反应是针对N-末端(NT)片段PCR。模板DNA 1 ng
      Oligo-F(50μM)
      1微升
      重叠-R(50μM)
      1微升
      10倍的HF缓冲液
      5微升
      dNTP(25μM)
      1微升
      Phusion高保真DNA聚合酶
      0.5微升
      H 2 0 
      添加到总共50微升
      注:退火温度为55-65°C,共28个循环。
    2. 对于C-末端(CT)片段PCR,使用oligo-R和重叠-F引物
    3. 使用QIAquick凝胶提取试剂盒凝胶纯化扩增的NT和CT片段
    4. 以1:1的比例混合纯化的片段作为新的模板DNA,并如下进行重叠PCR:
      混合模板DNA
      约100 ng
      10倍的HF缓冲液
      5微升
      dNTP(25μM)
      1微升
      Phusion高保真DNA聚合酶
      0.5微升
      H <2> O
      添加到总共50微升
    5. 运行10个循环,退火温度为55°C或更高。向反应体系中加入oligo-F(1μl)和oligo-R(1μl)后,继续运行额外的20个循环。
    6. 用凝胶纯化提取正确大小的片段
  2. 限制性内切酶体外消化
    DNA
    1微克
    10倍缓冲
    5微升
    限制性内切酶#1
    0.75微升
    限制性内切酶#2
    0.75微升
    H <2> O
    添加到总共50微升

    在37°C孵育2小时 注意:对于不同的酶,消化的最佳温度可能不同,例如BsmBI的55℃。
  3. 用限制性内切酶线性化载体并通过小牛肠碱性磷酸酶(CIP)去磷酸化
    1. 矢量线性化:与配方2相同
    2. 载体去磷酸化:在载体线性化后,加入1μlCIP,并在37℃温育40分钟。
  4. 体外DNA连接
    DNA插入
    12.5微升
    线性化矢量
    1微升
    10x T4 DNA连接酶缓冲液 1.5微升
    室温,1小时
    注意:阴性对照是通过用水替换DNA插入物而单独线性化载体的结扎。
  5. DNA转化
    1. 将连接产物与DH5α感受态细胞在冰上孵育30分钟
    2. 42°C热冲击1分钟
    3. 允许细胞在37℃下以220rpm的振荡速度恢复1小时,然后在含有适当抗生素的LB琼脂上铺板。
  6. 体外DNA寡核苷酸退火
    1. 将每种寡核苷酸溶于1x低EDTA TE缓冲液至50μM
    2. 混合等体积的退火寡核苷酸,并将它们置于95°C加热5分钟,然后将其关闭以缓慢冷却至室温。
  7. 体外T4多核苷酸激酶磷酸化
    退火的低聚物
    1微升
    10x T4 DNA连接酶缓冲液 1微升
    T4多核苷酸激酶
    1微升
    H <2> O
    添加到总共10微升

    在37°C孵育1小时 在-20°C储存
    注意:为了以后的DNA连接,磷酸化的寡核苷酸需要在使用前以1:500进行稀释。

致谢

我们感谢乔安娜•奥利里博士对本手稿的批判性校对。该项目得到了美国国立卫生研究院的RO3NS087416的支持。
利益冲突声明:作者声明不存在利益冲突或利益冲突。

参考

  1. Bodor,D.L.,Rodriguez,M.G.,Moreno,N.and Jansen,L.E。(2012)。 基于定量SNAP的脉冲追踪成像分析蛋白质周转率 Curr Protoc Cell Biol Chapter 8:Unit8 8.
  2. Esposito,D.,Garvey,L.A。和Chakiath,C.S。(2009)。 Gateway克隆蛋白表达的方法 Methods Mol Biol 498 :31-54。
  3. Santiago-Sim,T.,Fang,X.,Hennessy,ML,Nalbach,SV,DePalma,SR,Lee,MS,Greenway,SC,McDonough,B.,Hergenroeder,GW,Patek,KJ,Colosimo,SM,Qualmann ,KJ,Hagan,JP,Milewicz,DM,MacRae,CA,Dymecki,SM,Seidman,CE,Seidman,JG和Kim,DH(2016)。在颅内动脉瘤和蛛网膜下腔出血的发病机制中,THSD1(含血小板反应蛋白1型结构域的蛋白1)突变。 Stroke 47(12):3005-3013。
  4. Shaner,N.C。,Steinbach,P.A。和Tsien,R.Y。(2005)。 选择荧光蛋白的指南 Nat Methods 2 (12):905-909。
  5. Xu,Z.,Rui,Y.N.,Balzeau,J.,Menezes,M.R.,Niu,A.,Hagan,J.P.and Kim,D.H。(2017)。 通过IIS型限制性核酸内切酶介导的精确克隆进行的高效一步无瘢痕蛋白标记
  6. Xu,X.,Song,Y.,Li,Y.,Chang,J.,Zhang,H. and An,L.(2010)。 串联亲和纯化方法:用于蛋白质复合物纯化和蛋白质相互作用鉴定的高效系统
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引用:Xu, Z., Rui, Y., Hagan, J. P. and Kim, D. H. (2018). Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases. Bio-protocol 8(3): e2721. DOI: 10.21769/BioProtoc.2721.
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