参见作者原研究论文

本实验方案简略版
Dec 2018

本文章节


 

Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli
重组人胰脂肪酶在大肠杆菌中的表达和纯化   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an Escherichia coli (E. coli) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from the E. coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses against decreasing concentrations of urea in the presence of glycerol and Ca2+ for two days followed by gel filtration FPLC, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. Here we report the expression, purification, and optimized refolding procedures for active recHPL from E. coli, thus establishing it as a suitable system for the production of recHPL of high purity and scaling up.

Keywords: Human pancreatic lipase (人胰腺脂肪酶), Escherichia coli (大肠杆菌), Lipolytic activity (解脂活性), Refolding (复性), Strep-tag II (Strep-tag II), Gel filtration (凝胶过滤)

Background

Mammalian lipase, essential for the dietary fat digestion and absorption, can be used for development of an anti-obesity treatment. However, obtaining pure and stable native mammalian lipase has been a challenge because it is often degraded by co-existing proteases in the pancreatic juice. Due to the limited availability of human tissue samples and their potential risk for disease transmission, it may be best to prepare HPL using recombinant expression systems. Several methods have been reported to express recHPL using yeast (Yang and Lowe, 1998), insect (Thirstrup et al., 1993), or mammalian cells (Canalias et al., 1994), which require culturing for at least 5, 6, or 10 days, respectively. The recHPLs obtained using these cells require strict quality control to achieve homogeneity because they are differentially glycosylated. The use of E. coli as an expression system can avoid these problems in addition to providing other benefits such as ease of culture and purification. Therefore, we have established a protocol for optimized expression, purification, and refolding of active recHPL from E. coli. This method requires only two days of culturing, is easy to scale up at low cost, and is convenient because it circumvents the need to control the glycosylation pattern of the recombinant protein. Although the recHPL is non-glycosylated and native porcine pancreatic lipase (PPL) is glycosylated, both lipases show lipolytic activity equal to native PPL against tributyrin and triolein as substrates (Kawaguchi et al., 2018). It may be useful as an alternative to the PPL that has been used in the development of pharmaceutical products. For example, pancreatic enzyme replacement therapy for pancreatic exocrine insufficiency (Lowe and Whitcomb, 2015), and screening of inhibitor compounds to provide an important avenue for anti-obesity therapy (Seyedan et al., 2015). Other promising applications of recHPL are industrial biocatalysts for biotechnological processes (Sharma et al., 2001).

Materials and Reagents

  1. 50 ml tubes (Labcon, catalog number: 3181-345-008)
  2. 15 ml tubes (Labcon, catalog number: 3131-345-008)
  3. 1.5 ml tubes (BIO-BIK, catalog number: ST-0150F)
  4. 0.2 ml PCR tubes with Dome Cap (Axygen, Funakoshi, catalog number: PCR-02D-C)
  5. Pipette tips 10 μl (BIO-BIK, catalog number: 1050N), 200 μl (BIO-BIK, catalog number: PT-200), 1,000 μl (BIO-BIK, catalog number: 804C)
  6. 96-well microplates (Greiner Bio-One, catalog number: 655801)
  7. Disposable 2 ml polystyrene columns (Pierce, catalog number: 29920)
  8. Ultrafiltration discs, 10 kDa NMW (Millipore, catalog number: PBGC02510)
  9. Cellulose acetate membrane filter, 0.45 μm (Advantec, catalog number: C045A090C) 
  10. GL chromatodisc 0.45 μm (GL Science, 25N)
  11. PVDF membrane (Millipore, catalog number: IPVH00010)
  12. Dialysis membrane (As One, catalog number: 2-316-02)
  13. HiPrep 16/60 Sephacryl S-200 HR (GE Healthcare, catalog number: 17-1166-01)
  14. Sterile Petri dishes (As One, catalog number: 1-7484-01)
  15. E. coli DH5α competent cells (Takara Bio, catalog number: 9057)
  16. Stirred Ultrafiltration cells (Millipore, catalog number: 1532701)
  17. Primers (Invitrogen)
    Primer 1 (forward), 5′-AAAGAAGTTTGCTACGAAAGACTC-3′
    Primer 2 (reverse), 5′-GATTGTGCCACACTCCCACTCG-3′
    Primer 3 (forward), 5′-ACCAAAAGATGTCAACACCCGCTTCC-3′
    Primer 4 (reverse), 5′-GACCAAGGCAATATATGGAGGGGTC-3′
    Primer 5 (forward), 5′-ATGGTAGGTC TCAAATGAAAGAAGTTTGCTACGAAAGACTCG-3′
    Primer 6 (reverse), 5′-ATGGTAGGTCTCAGCGCTACACGGTGTGAGGGTGAGCAG-3′
    Primer 7 (forward), 5′-CCCAGCAGAACCTTGCTTTCAGG-3′
    Primer 8 (reverse), 5′-CTTCCCAGATTCCGTCTATGTCC-3′
  18. pASK-IBA3plus plasmid (IBA, catalog number: 2-1402-000)
  19. Strep-Tactin-Sepharose (IBA, catalog number: 2-1201-010)
  20. Human MTC Panel I (Clontech Laboratories, catalog number: 636742)
  21. PfuTurbo DNA Polymerase (Agilent Technologies, catalog number: 600250)
  22. KOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd., catalog number: SMK-101)
  23. MgSO4·7H2O (Wako, catalog number: 138-00415)
  24. Anti-lipase antibodies (Abcam, catalog number: ab96100)
  25. HRP-conjugated goat anti-rabbit antibodies (Kirkegaard and Perry Laboratories, catalog number: 074-15-061)
  26. BSA, Albumin from bovine serum, Low salt (Wako, 017-15124) 
  27. BsaI (BioLabs, catalog number: R0535)
  28. PCR purification kit (Wizard SV® Gel and PCR Clean-Up system) (Promega, catalog number: A9281) 
  29. Ligase enzyme T4 DNA Ligase (Toyobo Co. Ltd, Osaka)
  30. BigDye TerminatorTM Purification Kit (Thermo Fisher Scientific, catalog number: 4376484)
  31. FastDigest® Eco31I (Thermo Fisher Scientific, catalog number: FDO293)
  32. 10x FastDigest®Green Buffer (Thermo Fisher Scientific, catalog number: B72)
  33. Ligation high ver.2 (TOYOBO, catalog number: LGK-201)
  34. Ampicillin sodium (Wako, catalog number: 016-10373)
  35. Bacto Agar (Becton, Dickinson and Co., catalog number: 214010)
  36. Luria Bertani (LB) Broth Base (Invitrogen, catalog number: 12780-052)
  37. Anhydrotetracycline (IBA, catalog number: 2-0401-002)
  38. Urea (Wako, catalog number: 217-00171)
  39. Dithiothreitol (DTT) (Wako, catalog number: 049-08972)
  40. Desthiobiotin (Sigma-Aldrich, catalog number: D1411)
  41. Ethylenediaminetetraacetic acid (EDTA) (Wako, catalog number: 345-01865)
  42. Sodium chloride (NaCl) (Kanto Chemical Co., Inc., catalog number: 37144-86)
  43. Sodium hydroxide (NaOH) (Wako, catalog number: 198-13765)
  44. Phenylmethylsulfonyl fluoride (PMSF) (Nacalai Tesque, catalog number: 27327-94)
  45. Tris (hydroxylmethyl) aminomethane (Wako, catalog number: 204-07881)
  46. Triton X-100 (Sigma-Aldrich, catalog number: T9284)
  47. L-Arginine hydrochloride (Wako, catalog number: 018-04625)
  48. CBB R-250 (Wako, catalog number: 031-17922)
  49. Methanol (Wako, catalog number: 25183-70)
  50. Acetic acid (Wako, catalog number: 017-00251)
  51. L-Cystine dihydrochloride (Wako, catalog number: 03405322)
  52. L-Cysteine (Wako, catalog number: 039-20652)
  53. Glycerol (Wako, catalog number: 075-00611)
  54. CaCl2 (Wako, catalog number: 031-00435)
  55. Tributyrin (Wako, catalog number: 207-02392)
  56. Colipase, Pig (Bio-Rad, catalog number: 2148-1004)
  57. Sodium taurodeoxycholate
  58. LB agar plates (see Recipes)
  59. LB medium (see Recipes)
  60. Buffer A (see Recipes)
  61. Lysis buffer (see Recipes)
  62. Buffer A* (see Recipes)
  63. Equivalent buffer (see Recipes)
  64. Elution buffer (see Recipes)
  65. Refolding buffer (see Recipes)
  66. Running buffer (see Recipes)
  67. Reaction buffer (see Recipes)

Equipment

  1. 300 ml shaking flask (Iwaki, catalog number: 4070FK300)
  2. 500 ml glass beaker (e.g., As One, catalog number: 2-5091-06)
  3. Pipettes (Eppendorf, Reference 10-100 μl, 0.5-10 μl, catalog numbers: 4920000059, 4920000024)
  4. Magnetic stirring bar (NALGENE, catalog number: 93-5521-5)
  5. -80 °C freezer (Nihon Freezer MY BIO, model: VT-208)
  6. Zymoreactor II thermal cycler (ATTO) 
  7. Prism 3100 Avant sequencer (Applied Biosystems)
  8. Shaking incubator (e.g., Thomas, model: AT-12R)
  9. Refrigerated bench-top centrifuge (e.g., Eppendorf, model: 5417R)
  10. High speed centrifuge (Hitachi, model: CR20GIII)
  11. Sonicator (Branson, model: Advanced Sonifier 250)
  12. Spectrophotometer (DeNovix, model: DS-11)
  13. Microplate spectrophotometer (DS Pharma Biomedical, model: Viento XS)
  14. Fraction collector (e.g., Advantec, model: SF-2100)
  15. AmiconTM, Bioseparations Stirred Cell (Merck, catalog number: 8010)
  16. AKTA Purifier chromatography system (GE Healthcare)
  17. pH-stat AT-510 (Kyoto Electronics Manufacturing Co, Ltd.)

Software

  1. UNICORN (GE Healthcare), control software for the AKTA purifier chromatography system
  2. Gen 5 (Bio Tek), data collection and analysis software for the microplate reader
  3. AT-Win (Kyoto Electronics Manufacturing Co, Ltd.), the control software for the pH-stat, automatic potentiometric titrator, AT-510

Procedure

This procedure to prepare recHPL includes five steps: (A) construction of expression plasmid, (B) expression in E. coli, (C) isolation, (D) refolding, and (E) purification.

  1. Construction of HPL expression plasmid
    ST II-fused recHPL was cloned into the pASK-IBA3plus plasmid to allow expression in E. coli. The construct contains a C-terminal Strep-tag II to facilitate separation of recHPL (Figure 1).


    Figure 1. Sequence of recHPL. A. Part of the nucleotide sequence of an HPL-pASK-IBA3plus plasmid and the deduced amino acid sequence. The entire sequence of HPL was confirmed using an autosequencer. Boxed region: the coding sequence of HPL, wavy underlines: the sequence of the linker, dotted underlines: the sequence of the ST II-tag. B. Structure of pASK-IBA 3Plus-HPL plasmid.

    1. Following the PCR protocol detailed below, amplify the coding sequence of HPL using the human pancreas cDNA library, Human MTC Panel I as the template for PCR using a Zymoreactor II (ATTO) thermal cycler with following conditions:
      [Primers]
      Primer 1 (forward), 5′-AAAGAAGTTTGCTACGAAAGACTC-3′
      Primer 2 (reverse), 5′-GATTGTGCCACACTCCCACTCG-3′
      [Reaction mixture]

      [PCR conditions]
      30 cycles of
      95 °C
      60 s (Denaturation)

      55 °C
      60 s (Annealing)

      72 °C
      60 s (Extension)

    2. Separate the amplicon by agarose gel electrophoresis using 1% agarose gel.
    3. Cut out the DNA band (expected size 1,350 bp) on the gel and purify with Wizard® SV Gel and PCR Clean-Up System to obtain insert DNA.
    4. Perform a second PCR to replace the Eco311 restriction endonuclease recognition sequence GGTCTC contained in the insert DNA with GGTCAC using a Zymoreactor II (ATTO) thermal cycler with following conditions.
      [Primers]
      Primer 3 (forward), 5′-ACCAAAAGATGTCAACACCCGCTTCC-3′
      Primer 4 (reverse), 5′-GACCAAGGCAATATATGGAGGGGTC-3′ 

      [Reaction mixture]

      [PCR conditions]
      10 cycles of
      94 °C
      120 s

      98 °C
      10 s

      68 °C
      360 s

    5. Separate the amplicon by agarose gel electrophoresis using 1% agarose gel.
    6. Cut out the DNA band (expected size 1,350 bp) on the gel and purify it with Wizard® SV Gel and PCR Clean-Up System to obtain insert DNA.
    7. Perform a third PCR to obtain insert DNA with Eco31 I restriction endonuclease recognition site added at both ends using Zymoreactor II (ATTO) thermal cycler under the following condition:
      [Primers]
      Primer 5 (forward), 5′-ATGGTAGGTC TCAAATGAAAGAAGTTTGCTACGAAAGACTCG-3′
      Primer 6 (reverse), 5′-ATGGTAGGTCTCAGCGCTACACGGTGTGAGGGTGAGCAG-3′

      [Reaction mixture]

      [PCR conditions]
      Initial denaturation
      94 °C
      120 s
      25 cycles of
      94 °C
      15 s

      65 °C
      30 s

      68 °C
      60 s

    8. Purify the sequencing reactions using a BigDye TerminatorTM Purification Kit.
    9. Digest the amplicon 2 μl by adding FastDigest® Eco31I 1 μl, 10x FastDigest Green Buffer 2 μl, and ddH2O 15 μl at 37 °C for 5 min.
    10. Digest pASK-IBA3Plus 2 μl by adding FastDigest® Eco31I 1 μl, 10x FastDigest Green Buffer 2 μl, and ddH2O 15 μl at 37 °C for 5 min.
    11. Separate the digested samples by agarose gel electrophoresis using 1% agarose gel.
    12. Cut out the DNA bands (expected sizes of insert DNA and pASK-IBA3Plus are 1,350 bp and 3,247 bp, respectively) on the gel and purify with Wizard® SV Gel and PCR Clean-Up.
    13. Ligate the digested insert DNA with the digested pASK-IBA3plus to obtain an HPL-pASK-IBA3plus plasmid by DNA Ligation Kit, Ligation high Ver.2.
    14. Sequence all constructed plasmids using the Prism 3100 Avant sequencer to confirm the sequences of the designed recombinant HPLs. Use the following primers:
      Primer 7 (forward), 5′-CCCAGCAGAACCTTGCTTTCAGG-3′
      Primer 8 (reverse), 5′-CTTCCCAGATTCCGTCTATGTCC-3′

  2. Expression of recHPL in E. coli
    1. Transform 5 μl (10-fold dilution) of HPL-pASK-IBA3plus plasmid (ca. 2.5 ng) into 50 μl of E. coli DH5α competent cells in a 1.5 ml tube.
    2. Incubate for 30 min on ice.
    3. Incubate at 42 °C for 45 s.
    4. Add 900 μl of LB medium and culture at 37 °C for 1 h.
    5. Plate 100 μl of transformants on LB agar plates including 100 μg/ml ampicillin at 37 °C for 18 h.
    6. Pick up a single colony and grow the transformants in 5 ml LB medium including 100 μg/ml ampicillin in a 50 ml tube at 37 °C for 18 h.
    7. Transfer the bacterial suspension (3 ml) into 300 ml fresh LB medium including 100 μg/ml ampicillin in a shaking flask and culture for 3 h at 37 °C at 150 rpm.
    8. Add 30 μl of 2 mg/ml anhydrotetracycline at a final concentration of 200 μg/L when the absorbance of the culture measured at 550 nm by a spectrophotometer (DeNovix) has reached 0.5 to induce expression of recombinant protein.
    9. Incubate for 5 h at 30 °C with shaking at 120 rpm.
    10. Centrifuge at 6,100 x g for 15 min at 4 °C using a high speed centrifuge (Hitachi) to harvest cells.
    11. Discard the supernatant, resuspend the bacterial pellet in 15 ml of buffer A, and freeze at -80 °C in a 50 ml tube until use.

  3. Isolation of recHPL
    1. Thaw the frozen bacterial pellet.
    2. Lyse the bacterial cell pellet by sonication for 2 min (30 s x 4 times, output: 4, duty cycle: 70%) on ice. 
    3. Centrifuge at 4,400 x g for 15 min at 4 °C using a high speed centrifuge (Hitachi).
    4. Discard the supernatant and resuspend the pellet in 15 ml of lysis buffer.
    5. Centrifuge at 28,000 x g for 10 min at 4 °C.
    6. Discard the supernatant and wash the pellet twice with 30 ml of buffer A.
    7. Centrifuge at 28,000 x g for 10 min at 4 °C.
    8. Discard the supernatant and solubilize the washed inclusion bodies with 10 ml of buffer A* with agitation for 30 min at 4 °C. 
    9. Add 50 μl of 2 M DTT at a final concentration of 10 mM to the supernatant.
    10. Incubate for 1 h at 37 °C with agitation at 100 rpm.
    11. Dilute the solubilized proteins with 10 ml of buffer A*.
    12. Dialyze against 150 ml of the equivalent buffer using the dialysis membrane (pore diameter = 50 Å) at 4 °C for 2 h to remove urea in a glass beaker with gentle stirring using a magnetic stirrer.
    13. Centrifuge the dialyzed solution at 28,000 x g for 10 min at 4 °C. 
    14. Aspirate the supernatant and transfer it to a 50 ml tube.
    15. Add 20 ml of ice-cold equivalent buffer to the 2 ml of Strep-Tactin-Sepharose in a polystyrene column using a Pasteur pipette.
    16. Remove the equilibrated Strep-Tactin-Sepharose and add to the dialyzed protein solution in the 50 ml tube.
    17. Incubate at 4 °C for 30 min with gentle shaking at around 30 rpm.
    18. Pack the suspension mixture of Strep-Tactin-Sepharose into a polystyrene column.
    19. Wash with 20 ml of equivalent buffer at a flow rate of 3 drops/s, collecting 70-drop fractions using a fraction collector at 4 °C (Figure 2).
    20. Elute ST II-fused recHPL with elution buffer at a flow rate of 10 drops/s, collecting 22-drop fractions using a fraction collector at 4 °C.
    21. Measure the absorbance of 200 μl of fractions using 96-well microplates, a microplate spectrophotometer (DS Pharma Biomedical), and software Gen 5 at 280 nm at room temperature.
    22. Combine fractions with high A280 readings corresponding to the eluted protein and store the eluted peak fractions in a 15 ml tube at -80 °C. 


      Figure 2. Separation of recHPL by Strep-Tactin-Sepharose column. A. Affinity chromatography profile on a Strep-Tactin-Sepharose column (Schmidt et al., 1996). Elution was started from the fraction indicated by the arrow, and the peak was obtained at elution volume 19-26 ml. Column volume: 1.9 ml (i.e., 7 mm x 50 mm), flow rate: 10 s/drop, temperature: 4 °C. B. SDS-PAGE and Western blot analysis of the recHPL-ST II. From left lane, “CBB” shows a CBB-stained 12% polyacrylamide gel. “Anti-HPL” shows an electroblotted PVDF membrane blocked with 3% BSA and stained using the primary antibody, anti-lipase antibodies (1:3,000) at room temperature for 1 h and the secondary antibody, HRP-conjugated goat anti-rabbit antibodies (1:5,000) at room temperature for 1 h followed by developed using ECL reagent (GE Healthcare). 1, Marker proteins; 2, solubilized protein from inclusion body; 3 and 4, recHPL-ST II purified on the Strep-Tactin-Sepharose column.

  4. Refolding of RecHPL (Figure 3)


    Figure 3. Procedure for refolding of isolated recHPL 

    1. Thaw the frozen eluted peak fractions and measure the absorbance at 280 nm at room temperature.
    2. Add urea to a final concentration of 8 M to 7-12 ml of the purified 0.1-0.2 mg/ml recHPL in peak fractions.
    3. Add 2 M DTT to a final concentration of 10 mM.
    4. Incubate for 2 h at 37 °C with agitation at approximately 100 rpm.
    5. Dialyze against 300 ml of refolding buffer containing a step-wise decrease in urea concentration (6 M and 4 M) at 4 °C for 2 h.
    6. Dialyze against 300 ml of refolding buffer containing 2 M urea at 4 °C overnight.
    7. Dialyze against 300 ml of refolding buffer containing step-wise decrease in urea concentration (1 M and 0.5 M) at 4 °C for 2 h.
    8. Dialyze against 300 ml of refolding buffer containing 0 M urea at 4 °C for 1 h.
    9. Centrifuge at 21,000 x g for 5 min and collect the supernatant to remove protein aggregates at 4 °C.

  5. Purification of recHPL (Figure 4)
    Note: Store all reagents and carry out all procedures at 4 °C.
    1. Add the supernatant of the refolded recHPL to an AmiconTM Bioseparations Stirred Cell with an ultrafiltration disc (10 kDa NMWL).
    2. Concentrate recHPL by 2-10 times using the stirred cell.
    3. Filter the concentrated recHPL through the GL chromatodisc 0.45 μm (GL Science, 25N)
    4. Filter the running buffer for FPLC through a cellulose acetate membrane filter, 0.45 μm.
    5. Inject 1 ml of refolded recHPL into a HiPrep 16/60 Sephacryl S-200 HR column (column volume: 120 ml), pre-equilibrated with running buffer on an AKTA Purifier FPLC system at a flow rate of 0.5 ml/min.
    6. Elute the column with 180 ml of running buffer at a flow rate of 0.5 ml/min, collecting 2 ml fractions.
    7. Measure the absorbance of fractions at 280 nm and store approximately 5 fractions (each 2 ml) of the second peak as active recHPL.
    8. Determine the lipolytic activity of recHPL using tributyrin as substrate by automated titration of fatty acids released with 10 mM NaOH using the pH-stat AT-510 (Kawaguchi et al., 2018).


      Figure 4. Purification of active recHPL by gel-filtration FPLC. Elution profile of recHPL on size exclusion. RecHPL after refolding was loaded onto a HiPrep 16/60 Sephacryl S-200 column (column volume: 120 ml) and eluted using 10 mM TBS containing 400 mM L-Arg at a flow rate of 0.5 ml/min at 4 °C. Protein peaks were detected by measuring absorbance at 280 nm using a UPC-900 monitor and AKTA Purifier. Active recHPL-ST II (1.8-6 mg) was obtained from 1 L of culture, and it showed a specific activity of 1.2-1.9 kU/mg. Specific activity of the fractions was measured using 0.11 M tributyrin emulsified in 1 ml reaction buffer as the substrate. The measurements were performed containing colipase in excess, 0.5 μg/50 μl. After addition of each fraction eluted by gel filtration FPLC in Figure 4 (0.5 μg/50 μl), the NaOH consumption was recorded at a reaction temperature of 37 °C using the automatic potentiometric titrator, pH-stat AT-510 (Kyoto Electronics Manufacturing Co. Ltd.). One unit was defined as the amount of enzyme that released 1.0 μmol of free fatty acid per min.

Data analysis

Protein expression is verified by Coomassie staining and Western blotting using anti-lipase antibodies (Abcam, ab96100). Chromatograms were recorded using UNICORN software. Gen 5 software was used for data collection and analysis for the microplate reader. The pH-stat AT-510 was controlled by AT-Win software.

Notes

  1. Prepare 0.2 M PMSF stock solution in ethanol and dilute to buffer just before use.
  2. In a comparison of TBS supplemented with 8 M urea, 1% Triton X-100, or 1% SDS, TBS containing 8 M urea is the most efficient method of solubilizing recHPL-ST II from E. coli lysates.
  3. A Strep-Tactin-Sepharose column is an open column using gravity to move the solvent. The flow rate is controlled by the hydrostatic pressure on top of the column and the stopcock at the bottom of the column.
  4. All the processes for isolation, dialysis for refolding, and purification of recHPL should be performed at 4 °C except for the denaturation processes with 8 M urea and 10 mM DTT, which must be done at 37 °C. 
  5. The addition of L-Arg showed a remarkable effect on the refolding of recHPL-ST II, resulting in a high yield, which must be attributable to the inhibition of aggregation of recHPL-ST II by L-Arg.

Recipes

  1. LB agar plates
    20 g/L LB Broth Base
    15 g/L bacto agar
    Autoclave at 121 °C, 2.6 bar for 20 min to sterilize
    Add ampicillin (100 μg/ml) and pour the medium into sterile Petri dishes
  2. LB medium
    20 g/L LB Broth Base
    Autoclave at 121 °C, 2.6 bar for 20 min to sterilize
  3. Buffer A
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    1 mM EDTA
  4. Lysis buffer
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    1 mM EDTA
    0.5% Triton X-100
  5. Buffer A*
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    1 mM EDTA
    8 M urea
    1 mM PMSF 
  6. Equivalent buffer
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    400 mM L-Arg
    1 mM EDTA
    1 mM PMSF
  7. Elution buffer
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    400 mM L-Arg
    1 mM EDTA
    1 mM PMSF
    2.5 mM desthiobiotin
  8. Refolding buffer
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    10% glycerol
    2.5 mM CaCl2
    1 mM PMSF
    1 mM cysteine
    0.1 mM cystine
    400 mM L-Arg
    Varing urea concentration: 6 M, 4 M, 2 M, 1 M, 0.5 M, and 0 M urea
  9. Running buffer
    10 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    400 mM L-Arg
  10. Reaction buffer
    1 mM Tris-HCl (pH 7.5)
    4 mM sodium taurodeoxycholate
    100 mM NaCl
    5 mM CaCl2

Acknowledgments

We sincerely thank Daiwa Securities Health Foundation (HO) and the Japan Foundation of Applied Enzymology (HO) for support with research funds. We sincerely thank Prof. Yusuke Suzuki, Ms. Chihiro Tomita, Ms. Rina Naradate, Ms. Tomoko Higami, Dr. Kosuke Nakamura, and Ms. Hiromi Sakagami for assisting in this work. We thank Mrs. K. Ono for English editing.

Competing interests

The authors declare there are no conflicts of interest.

References

  1. Canalias, F., Visvikis, A., Thioudellet, C. and Siest, G. (1994). Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme. Clin Chem 40(7 Pt 1): 1251-1257.
  2. Kawaguchi, N., Date, K., Suzuki, Y., Tomita, C., Naradate, R., Higami, T., Nakamura, K., Aikawa, K. and Ogawa, H. (2018). A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli. J Biochem 164(6): 407-414.
  3. Lowe, M. E. and Whitcomb, D. C. (2015). Next generation of pancreatic enzyme replacement therapy: Recombinant microbial enzymes and finding the perfect lipase. Gastroenterology 149(7): 1678-1681.
  4. Schmidt, G., Koepke, J., Frank, R. and Skerra, A. (1996). Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol 255: 753-766.
  5. Seyedan, A., Alshawsh, M. A., Alshagga, M. A., Koosha, S. and Mohamed, Z. (2015). Medicinal plants and their inhibitory activities against pancreatic lipase: A review. Evid Based Complement Alternat Med 2015: 973143.
  6. Sharma, R., Chisti, Y. and Banerjee, C. (2001). Production, purification, characterization, and applications of lipases. Biotechnol Adv 19: 627-662.
  7. Thirstrup, K., Carriere, F., Hjorth, S., Rasmussen, P. B., Woldike, H., Nielsen, P. F. and Thim, L. (1993). One-step purification and characterization of human pancreatic lipase expressed in insect cells. FEBS Lett 327(1): 79-84.
  8. Yang, Y. and Lowe, M. E. (1998). Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization. Protein Expr Purif 13(1): 36-40.

简介

人胰脂肪酶(HPL)是参与饮食脂肪消化的主要脂肪分解酶。 使用短Strep-tag II(STII)在大肠杆菌(E.coli)表达系统中首次成功制备了活性重组人胰脂肪酶(recHPL)。 将recHPL-ST II用来自大肠杆菌裂解物的8M尿素溶解,并在Strep-Tactin-Sepharose柱上纯化。 在甘油和Ca 2+ + 存在下通过逐步透析降低尿素浓度两天后重新折叠,然后进行凝胶过滤FPLC,从1L获得1.8-6mg活性recHPL-ST II。 文化。 在这里,我们报告来自 E的活性recHPL的表达,纯化和优化的重折叠程序。 大肠杆菌,从而将其建立为生产高纯度和扩大规模的recHPL的合适系统。
【背景】对膳食脂肪消化和吸收必不可少的哺乳动物脂肪酶可用于开发抗肥胖治疗。然而,获得纯的和稳定的天然哺乳动物脂肪酶一直是一个挑战,因为它经常被胰液中共存的蛋白酶降解。由于人体组织样本的有限性及其潜在的疾病传播风险,最好使用重组表达系统制备HPL。已经报道了几种使用酵母(Yang和Lowe,1998),昆虫(Thirstrup 等人,1993)或哺乳动物细胞(Canalias 等人)来表达recHPL的方法。 ,1994),分别需要培养至少5天,6天或10天。使用这些细胞获得的recHPL需要严格的质量控制以实现均一性,因为它们是差异糖基化的。使用 E。作为表达系统的大肠杆菌除了提供其他益处如易于培养和纯化之外,还可以避免这些问题。因此,我们已经建立了一个协议,用于优化来自 E的活性recHPL的表达,纯化和重折叠。这种方法仅需要两天的培养,易于按比例放大,并且方便,因为它不需要控制重组蛋白的糖基化模式。虽然recHPL是非糖基化的并且天然猪胰脂肪酶(PPL)是糖基化的,但两种脂肪酶都显示出与针对三丁酸甘油酯和三油精作为底物的天然PPL相等的脂解活性(Kawaguchi等,等,2018)。它可用作PPL的替代物,其已用于药物产品的开发。例如,用于胰腺外分泌功能不全的胰酶替代疗法(Lowe和Whitcomb,2015),以及筛选抑制剂化合物以提供抗肥胖治疗的重要途径(Seyedan et al。,2015)。 recHPL的其他有希望的应用是用于生物技术过程的工业生物催化剂(Sharma 等人,,2001)。

关键字:人胰腺脂肪酶, 大肠杆菌, 解脂活性, 复性, Strep-tag II, 凝胶过滤

材料和试剂

  1. 50毫升管(Labcon,目录号:3181-345-008)
  2. 15毫升管(Labcon,目录号:3131-345-008)
  3. 1.5毫升管(BIO-BIK,目录号:ST-0150F)
  4. 带有圆顶帽的0.2 ml PCR管(Axygen,Funakoshi,目录号:PCR-02D-C)
  5. 移液器吸头10μl(BIO-BIK,目录号:1050N),200μl(BIO-BIK,目录号:PT-200),1,000μl(BIO-BIK,目录号:804C)
  6. 96孔微孔板(Greiner Bio-One,目录号:655801)
  7. 一次性2毫升聚苯乙烯柱(Pierce,目录号:29920)
  8. 超滤盘,10 kDa NMW(Millipore,目录号:PBGC02510)
  9. 醋酸纤维素膜过滤器,0.45μm(Advantec,目录号:C045A090C) 
  10. GL chromatodisc0.45μm(GL Science,25N)
  11. PVDF膜(Millipore,目录号:IPVH00010)
  12. 透析膜(As One,目录号:2-316-02)
  13. HiPrep 16/60 Sephacryl S-200 HR(GE Healthcare,目录号:17-1166-01)
  14. 无菌培养皿(As One,目录号:1-7484-01)
  15. 电子。大肠杆菌DH5α感受态细胞(Takara Bio,目录号:9057)
  16. Stirred Ultrafiltration cells(Millipore,目录号:1532701)
  17. 引物(Invitrogen)
    引物1(正向),5'-AAAGAAGTTTGCTACGAAAGACTC-3'
    引物2(反向),5'-GATTGTGCCACACTCCCACTCG-3'
    引物3(前向),5'-ACCAAAAGATGTCAACACCCGCTTCC-3'
    引物4(反向),5'-GACCAAGGCAATATATGGAGGGGTC-3'
    引物5(前向),5'-ATGGTAGGTC TCAAATGAAAGAAGTTTGCTACGAAAGACTCG-3'
    引物6(反向),5'-ATGGTAGGTCTCAGCGCTACACGGTGTGAGGGTGAGCAG-3'
    引物7(前向),5'-CCCAGCAGAACCTTGCTTTCAGG-3'
    引物8(反向),5'-CTTCCCAGATTCCGTCTATGTCC-3'
  18. pASK-IBA3plus质粒(IBA,目录号:2-1402-000)
  19. Strep-Tactin-Sepharose(IBA,目录号:2-1201-010)
  20. Human MTC Panel I(Clontech Laboratories,目录号:636742)
  21. PfuTurbo DNA聚合酶(安捷伦科技,目录号:600250)
  22. KOD-Plus-Mutagenesis Kit(Toyobo Co.,Ltd。,目录号:SMK-101)
  23. MgSO 4 ·7H 2 O(Wako,目录号:138-00415)
  24. 抗脂肪酶抗体(艾博抗(上海)贸易有限公司,目录号:ab96100)
  25. HRP缀合的山羊抗兔抗体(Kirkegaard和Perry Laboratories,目录号:074-15-061)
  26. BSA,牛血清白蛋白,低盐(Wako,017-15124) 
  27. BsaI(BioLabs,目录号:R0535)
  28. PCR纯化试剂盒(Wizard SV ®凝胶和PCR Clean-Up系统)(Promega,目录号:A9281) 
  29. 连接酶T4 DNA连接酶(Toyobo Co. Ltd,Osaka)
  30. BigDye Terminator TM 纯化试剂盒(Thermo Fisher Scientific,目录号:4376484)
  31. FastDigest ® Eco31I(赛默飞世尔科技,目录号:FDO293)
  32. 10x FastDigest ®绿色缓冲液(Thermo Fisher Scientific,目录号:B72)
  33. 结扎高ver.2(TOYOBO,目录号:LGK-201)
  34. 氨苄西林钠(Wako,目录号:016-10373)
  35. Bacto Agar(Becton,Dickinson and Co.,目录号:214010)
  36. Luria Bertani(LB)肉汤基(Invitrogen,目录号:12780-052)
  37. 无水四环素(IBA,目录号:2-0401-002)
  38. 尿素(Wako,目录号:217-00171)
  39. 二硫苏糖醇(DTT)(Wako,目录号:049-08972)
  40. Desthiobiotin(Sigma-Aldrich,目录号:D1411)
  41. 乙二胺四乙酸(EDTA)(Wako,目录号:345-01865)
  42. 氯化钠(NaCl)(Kanto Chemical Co.,Inc。,目录号:37144-86)
  43. 氢氧化钠(NaOH)(Wako,目录号:198-13765)
  44. 苯甲基磺酰氟(PMSF)(Nacalai Tesque,目录号:27327-94)
  45. 三(羟甲基)氨基甲烷(Wako,目录号:204-07881)
  46. Triton X-100(Sigma-Aldrich,目录号:T9284)
  47. L-精氨酸盐酸盐(Wako,目录号:018-04625)
  48. CBB R-250(Wako,目录号:031-17922)
  49. 甲醇(Wako,目录号:25183-70)
  50. 乙酸(Wako,目录号:017-00251)
  51. L-胱氨酸二盐酸盐(Wako,目录号:03405322)
  52. L-半胱氨酸(Wako,目录号:039-20652)
  53. 甘油(Wako,目录号:075-00611)
  54. CaCl 2 (Wako,目录号:031-00435)
  55. Tributyrin(Wako,目录号:207-02392)
  56. 脂肪酶,猪(Bio-Rad,目录号:2148-1004)
  57. 牛磺酸脱氧胆酸钠
  58. LB琼脂平板(见食谱)
  59. LB培养基(见食谱)
  60. 缓冲液A(见食谱)
  61. 裂解缓冲液(见食谱)
  62. 缓冲液A *(见食谱)
  63. 等效缓冲液(见食谱)
  64. 洗脱缓冲液(见食谱)
  65. 重折叠缓冲液(见食谱)
  66. 运行缓冲区(参见食谱)
  67. 反应缓冲液(见食谱)

设备

  1. 300毫升摇瓶(Iwaki,目录号:4070FK300)
  2. 500毫升玻璃烧杯(例如,As One,目录号:2-5091-06)
  3. 移液器(Eppendorf,参考10-100μl,0.5-10μl,目录号:4920000059,4920000024)
  4. 磁力搅拌棒(NALGENE,目录号:93-5521-5)
  5. -80°C冰箱(Nihon Freezer MY BIO,型号:VT-208)
  6. Zymoreactor II热循环仪(ATTO) 
  7. Prism 3100 Avant测序仪(Applied Biosystems)
  8. 摇动培养箱(例如,Thomas,型号:AT-12R)
  9. 冷藏台式离心机(例如,Eppendorf,型号:5417R)
  10. 高速离心机(日立,型号:CR20GIII)
  11. Sonicator(Branson,型号:Advanced Sonifier 250)
  12. 分光光度计(DeNovix,型号:DS-11)
  13. 微孔板分光光度计(DS Pharma Biomedical,型号:Viento XS)
  14. 馏分收集器(例如,Advantec,型号:SF-2100)
  15. Amicon TM ,Bioseparations Stirred Cell(Merck,目录号:8010)
  16. AKTA净化器色谱系统(GE Healthcare)
  17. pH-stat AT-510(京都电子制造有限公司)

软件

  1. UNICORN(GE Healthcare),AKTA净化器色谱系统的控制软件
  2. Gen 5(Bio Tek),用于酶标仪的数据收集和分析软件
  3. AT-Win(京都电子制造有限公司),用于pH-stat的控制软件,自动电位滴定仪,AT-510

程序

制备recHPL的该方法包括五个步骤:(A)表达质粒的构建,(B)在 E中的表达。大肠杆菌,(C)分离,(D)重折叠和(E)纯化。

  1. HPL表达质粒的构建
    将ST II融合的recHPL克隆到pASK-IBA3plus质粒中以允许在 E中表达。大肠杆菌该构建体含有C末端Strep-tag II以促进recHPL的分离(图1)。


    图1. recHPL的序列。 :一种。 HPL-pASK-IBA3plus质粒的部分核苷酸序列和推导的氨基酸序列。使用自动测序仪确认HPL的整个序列。盒装区域: HPL 的编码序列,波浪下划线:连接子序列,虚线下划线:ST II标签的序列。 B.pASK-IBA 3Plus-HPL质粒的结构。

    1. 按照下面详述的PCR方案,使用人胰腺cDNA文库,人MTC Panel I作为PCR的模板,使用Zymoreactor II(ATTO)热循环仪扩增HPL的编码序列,条件如下:
      [引子]
      引物1(正向),5'-AAAGAAGTTTGCTACGAAAGACTC-3'
      引物2(反向),5'-GATTGTGCCACACTCCCACTCG-3'
      [反应混合物]

      [PCR条件] class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>
      的30个周期 95°C
      60秒(变性)

      55°C
      60秒(退火)

      72°C
      60秒(续)

    2. 使用1%琼脂糖凝胶通过琼脂糖凝胶电泳分离扩增子。
    3. 切下凝胶上的DNA条带(预期大小为1,350 bp),用Wizard ® SV凝胶和PCR Clean-Up System纯化以获得插入DNA。
    4. 使用Zymoreactor II(ATTO)热循环仪在以下条件下用GGTCAC替换插入DNA中包含的Eco311限制性内切核酸酶识别序列GGTCTC进行第二次PCR。
      [引子]
      引物3(前向),5'-ACCAAAAGATGTCAACACCCGCTTCC-3'
      引物4(反向),5'-GACCAAGGCAATATATGGAGGGGTC-3' 

      [反应混合物]

      [PCR条件] class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>
      的10个周期 94°C
      120 s

      98°C
      10秒

      68°C
      360 s

    5. 使用1%琼脂糖凝胶通过琼脂糖凝胶电泳分离扩增子。
    6. 切下凝胶上的DNA条带(预期大小为1,350 bp),用Wizard ® SV Gel和PCR Clean-Up System纯化,得到插入DNA。
    7. 进行第三次PCR以获得插入DNA,在以下条件下使用Zymoreactor II(ATTO)热循环仪在两端添加Eco31 I限制性内切核酸酶识别位点:
      [引子]
      引物5(前向),5'-ATGGTAGGTC TCAAATGAAAGAAGTTTGCTACGAAAGACTCG-3'
      引物6(反向),5'-ATGGTAGGTCTCAGCGCTACACGGTGTGAGGGTGAGCAG-3'

      [反应混合物]

      [PCR条件] class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>初期变性
      94°C
      120 s

      的25个周期 94°C
      15 s

      65°C
      30秒

      68°C
      60秒

    8. 使用BigDye Terminator TM 纯化试剂盒纯化测序反应。
    9. 通过在37°C下加入FastDigest ®Eco31I1μl,10x FastDigest Green Buffer2μl和ddH 2 O15μl消化扩增子2μl,持续5 min。
    10. 通过在37℃下加入FastDigest ®Eco31I1μl,10x FastDigest Green Buffer2μl和ddH 2 O15μl消化pASK-IBA3Plus2μl,持续5分钟。
    11. 使用1%琼脂糖凝胶通过琼脂糖凝胶电泳分离消化的样品。
    12. 在凝胶上切下DNA条带(插入DNA和pASK-IBA3Plus的预期大小分别为1,350 bp和3,247 bp),并用Wizard ® SV Gel和PCR Clean-Up进行纯化。
    13. 用消化的pASK-IBA3plus连接消化的插入DNA,通过DNA连接试剂盒,Ligation high Ver.2获得HPL-pASK-IBA3plus质粒。
    14. 使用Prism 3100 Avant测序仪对所有构建的质粒进行测序,以确认设计的重组HPL的序列。使用以下引物:
      引物7(前向),5'-CCCAGCAGAACCTTGCTTTCAGG-3'
      引物8(反向),5'-CTTCCCAGATTCCGTCTATGTCC-3'

  2. recHPL在 E中的表达。大肠杆菌
    1. 将5μl(10倍稀释)HPL-pASK-IBA3plus质粒( ca。 2.5ng)转化到50μl E中。大肠杆菌DH5α感受态细胞在1.5ml管中。
    2. 在冰上孵育30分钟。
    3. 在42°C孵育45秒。
    4. 加入900μlLB培养基并在37°C培养1小时。
    5. 将包含100μg/ ml氨苄青霉素的LB琼脂平板上的100μl转化体在37℃下平板培养18小时。
    6. 挑取单个菌落,将转化体在含有100μg/ ml氨苄青霉素的5ml LB培养基中于50ml管中于37℃培养18小时。
    7. 将细菌悬浮液(3ml)转移到摇瓶中的包含100μg/ ml氨苄青霉素的300ml新鲜LB培养基中,并在37℃,150rpm下培养3小时。
    8. 当通过分光光度计(DeNovix)在550nm处测量的培养物的吸光度达到0.5以诱导重组蛋白的表达时,添加30μl的2mg / ml无水四环素,终浓度为200μg/ L.
    9. 在30℃下以120rpm振荡孵育5小时。
    10. 使用高速离心机(Hitachi)在4℃下以6,100 x g 离心15分钟以收获细胞。
    11. 弃去上清液,将细菌沉淀重悬于15 ml缓冲液A中,并在-80°C下在50 ml管中冷冻直至使用。

  3. 隔离recHPL
    1. 解冻冷冻的细菌颗粒。
    2. 在冰上通过超声处理裂解细菌细胞沉淀2分钟(30秒×4次,输出:4,工作循环:70%)。 
    3. 使用高速离心机(Hitachi)在4℃下以4,400 x g 离心15分钟。
    4. 弃去上清液,将沉淀重悬于15ml裂解缓冲液中。
    5. 在4℃下以28,000 x g 离心10分钟。
    6. 弃去上清液,用30ml缓冲液A洗涤沉淀两次。
    7. 在4℃下以28,000 x g 离心10分钟。
    8. 弃去上清液,用10ml缓冲液A *溶解洗涤的包涵体,在4℃搅拌30分钟。
    9. 向上清液中加入50μl终浓度为10mM的2M DTT。
    10. 在37°C孵育1小时,同时以100 rpm的速度搅拌。
    11. 用10ml缓冲液A *稀释溶解的蛋白质。
    12. 使用透析膜(孔径= 50)在4℃下对150ml等效缓冲液透析2小时以在玻璃烧杯中使用磁力搅拌器在温和搅拌下除去尿素。
    13. 将透析的溶液在28,000 下在4℃下离心10分钟。 
    14. 吸出上清液并转移至50ml管中。
    15. 使用巴斯德吸管在聚苯乙烯柱中向2ml Strep-Tactin-Sepharose中加入20ml冰冷的等效缓冲液。
    16. 除去平衡的Strep-Tactin-Sepharose并加入50ml管中透析的蛋白质溶液中。
    17. 在4°C孵育30分钟,在约30转/分的温度下轻轻摇动。
    18. 将Strep-Tactin-Sepharose的悬浮液混合物装入聚苯乙烯柱中。
    19. 用3ml / s的流速用20ml等效缓冲液洗涤,使用馏分收集器在4℃收集70滴馏分(图2)。
    20. 使用洗脱缓冲液以10滴/秒的流速洗脱ST II熔融的recHPL,使用馏分收集器在4℃下收集22滴馏分。
    21. 使用96孔微量培养板,微孔板分光光度计(DS Pharma Biomedical)和软件Gen 5在室温下在280nm处测量200μl级分的吸光度。
    22. 将具有对应于洗脱蛋白的高A280读数的级分合并,并将洗脱的峰级分保存在-80℃的15ml管中。 


      图2.通过Strep-Tactin-Sepharose柱分离recHPL。 A.在Strep-Tactin-Sepharose柱上的亲和层析谱(Schmidt 等,。,1996)。从箭头所示的级分开始洗脱,并在洗脱体积19-26ml下获得峰。柱体积:1.9ml(即,7mm×50mm),流速:10s / drop,温度:4℃。 B.RecHPL-ST II的SDS-PAGE和Western印迹分析。从左侧泳道,“CBB”显示CBB染色的12%聚丙烯酰胺凝胶。 “抗HPL”显示用3%BSA封闭的电印迹PVDF膜,并使用一抗,抗脂肪酶抗体(1:3,000)在室温下染色1小时,并且二抗,HRP缀合的山羊抗兔抗体(1:5,000)在室温下1小时,然后使用ECL试剂(GE Healthcare)显色。 1,标记蛋白质; 2,来自包涵体的溶解蛋白质; 3和4,在Strep-Tactin-Sepharose柱上纯化的recHPL-ST II。

  4. RecHPL的重新折叠(图3)


    图3.重新分离recHPL的程序 

    1. 解冻冷冻洗脱的峰级分并在室温下测量280nm处的吸光度。
    2. 在峰值级分中加入尿素至终浓度为8M至7-12ml的纯化的0.1-0.2mg / ml recHPL。
    3. 加入2 M DTT至终浓度为10 mM。
    4. 在37°C孵育2小时,同时以约100 rpm的速度搅拌。
    5. 对300ml重折叠缓冲液透析,该缓冲液含有在4℃下逐步降低尿素浓度(6M和4M)2小时。
    6. 在4℃下对300ml含有2M尿素的重折叠缓冲液透析过夜。
    7. 对300ml重折叠缓冲液进行透析,该缓冲液含有在4℃下逐步降低尿素浓度(1M和0.5M)2小时。
    8. 在4℃下对含有0M尿素的300ml重折叠缓冲液透析1小时。
    9. 在21,000 x g 离心5分钟,收集上清液,在4°C下去除蛋白质聚集体。

  5. recHPL的纯化(图4)
    注意:储存所有试剂并在4°C下执行所有程序。
    1. 将重折叠的recHPL的上清液加入到具有超滤盘(10kDa NMWL)的Amicon TM Bioseparations Stirred Cell中。
    2. 使用搅拌的细胞将recHPL浓缩2-10倍。
    3. 通过GL chromatodisc0.45μm过滤浓缩的recHPL(GL Science,25N)
    4. 通过0.45μm的醋酸纤维素膜过滤器过滤FPLC的运行缓冲液。
    5. 将1ml重折叠的recHPL注入HiPrep 16/60 Sephacryl S-200HR柱(柱体积:120ml)中,用AKTA Purifier FPLC系统上的运行缓冲液以0.5ml / min的流速预平衡。
    6. 用180ml运行缓冲液以0.5ml / min的流速洗脱柱子,收集2ml级分。
    7. 测量280nm处级分的吸光度,并储存约5个级分(每2ml)的第二个峰作为活性recHPL。
    8. 使用pH-stat AT-510自动滴定用10mM NaOH释放的脂肪酸,使用三丁酸甘油酯作为底物确定recHPL的脂解活性(Kawaguchi 等人,2018)。


      图4.通过凝胶过滤FPLC纯化活性recHPL。 recHPL在尺寸排阻方面的洗脱曲线。将重折叠后的RecHPL加载到HiPrep 16/60 Sephacryl S-200柱(柱体积:120ml)上,并使用含有400mM L-Arg的10mM TBS以0.5ml / min的流速在4℃洗脱。通过使用UPC-900监测器和AKTA纯化器测量280nm处的吸光度来检测蛋白质峰。从1L培养物中获得活性recHPL-ST II(1.8-6mg),其比活性为1.2-1.9kU / mg。使用在1ml反应缓冲液中乳化的0.11M三丁酸甘油酯作为底物测量级分的比活性。进行含有过量,0.5μg/50μl的脂肪酶的测量。添加通过图4中的凝胶过滤FPLC(0.5μg/50μl)洗脱的各级分后,使用自动电位滴定仪pH-stat AT-510(Kyoto Electronics Manufacturing)在37℃的反应温度下记录NaOH消耗量。有限公司。)。一个单位定义为每分钟释放1.0μmol游离脂肪酸的酶量。

数据分析

通过考马斯染色和使用抗脂肪酶抗体(Abcam,ab96100)的蛋白质印迹验证蛋白质表达。使用UNICORN软件记录色谱图。 Gen 5软件用于酶标仪的数据收集和分析。 pH-stat AT-510由AT-Win软件控制。

笔记

  1. 在乙醇中制备0.2M PMSF储备溶液,并在使用前稀释至缓冲液。
  2. 在补充有8M尿素,1%Triton X-100或1%SDS的TBS的比较中,含有8M尿素的TBS是从 E溶解recHPL-ST II的最有效方法。大肠杆菌裂解液。
  3. Strep-Tactin-Sepharose柱是使用重力移动溶剂的开放柱。流速由色谱柱顶部的静水压力和色谱柱底部的活塞控制。
  4. 除了8M尿素和10mM DTT的变性过程外,所有分离,重折叠透析和recHPL纯化的过程都应在4℃下进行,这必须在37℃下进行。 
  5. 加入L-Arg对recHPL-ST II的重折叠显示出显着的效果,导致高产率,这必须归因于L-Arg抑制recHPL-ST II的聚集。

食谱

  1. LB琼脂平板
    20克/升LB肉汤基地
    15克/升bacto琼脂
    高压灭菌器在121°C,2.6巴下进行20分钟灭菌 加入氨苄青霉素(100μg/ ml),将培养基倒入无菌培养皿中
  2. LB媒体
    20克/升LB肉汤基地
    高压灭菌器在121°C,2.6巴下进行20分钟灭菌
  3. 缓冲区A
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    1 mM EDTA
  4. 裂解缓冲液
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    1 mM EDTA
    0.5%Triton X-100
  5. 缓冲区A *
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    1 mM EDTA
    8 M尿素
    1 mM PMSF 
  6. 等效缓冲区
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    400 mM L-Arg
    1 mM EDTA
    1 mM PMSF
  7. 洗脱缓冲液
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    400 mM L-Arg
    1 mM EDTA
    1 mM PMSF
    2.5mM脱硫生物素
  8. 重塑缓冲区
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    10%甘油
    2.5 mM CaCl 2
    1 mM PMSF
    1 mM半胱氨酸
    0.1 mM胱氨酸
    400 mM L-Arg
    尿素浓度:6M,4M,2M,1M,0.5M和0M尿素
  9. 运行缓冲区
    10mM Tris-HCl(pH 8.0)
    150 mM NaCl
    400mM L-Arg
  10. 反应缓冲液
    1 mM Tris-HCl(pH 7.5)
    4 mM牛磺酸脱氧胆酸钠
    100 mM NaCl
    5mM CaCl 2,

致谢

我们衷心感谢大和证券健康基金会(HO)和日本应用酶学基金会(HO)对研究基金的支持。我们衷心感谢Suusuki Yusuke教授,Chihiro Tomita女士,Rina Naradate女士,Tomoko Higami女士,中村康介博士和Hiromi Sakagami女士协助这项工作。我们感谢K. Ono女士的英文编辑。

利益争夺

作者声明没有利益冲突。

参考

  1. Canalias,F.,Visvikis,A.,Thioudellet,C。和Siest,G。(1994)。 V79细胞中酶活性人胰脂肪酶的稳定表达:重组酶的纯化和表征。 / a> Clin Chem 40(7 Pt 1):1251-1257。
  2. Kawaguchi,N.,Date,K.,Suzuki,Y.,Tomita,C.,Naradate,R.,Higami,T.,Nakamura,K.,Aikawa,K。和Ogawa,H。(2018)。 从大肠杆菌中制备活性重组人胰脂肪酶的新方案。 J Biochem 164(6):407-414。
  3. Lowe,M。E.和Whitcomb,D。C.(2015)。 下一代胰酶替代疗法:重组微生物酶,寻找完美的脂肪酶。 Gastroenterology 149(7):1678-1681。
  4. Schmidt,G.,Koepke,J.,Frank,R。和Skerra,A。(1996)。 Strep-tag亲和肽与其同源靶标链霉抗生物素蛋白之间的分子相互作用。 < em> J Mol Biol 255:753-766。
  5. Seyedan,A.,Alshawsh,M。A.,Alshagga,M。A.,Koosha,S。和Mohamed,Z。(2015)。 药用植物及其对胰脂肪酶的抑制活性:综述。 Evid基于补充Alternat Med 2015:973143。
  6. Sharma,R.,Chisti,Y。和Banerjee,C。(2001)。 脂肪酶的生产,纯化,表征和应用。 Biotechnol Adv 19:627-662。
  7. Thirstrup,K.,Carriere,F.,Hjorth,S.,Rasmussen,P.B.,Woldike,H.,Nielsen,P.F。和Thim,L。(1993)。 昆虫细胞中表达的人胰腺脂肪酶的一步纯化和表征。 FEBS Lett 327(1):79-84。
  8. Yang,Y。和Lowe,M。E.(1998)。 在酵母细胞中表达的人胰腺甘油三酯脂肪酶:纯化和表征。 蛋白质Expr Purif 13(1):36-40。
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: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kawaguchi, N., Ogawa, H. and Date, K. (2019). Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli. Bio-protocol 9(13): e3286. DOI: 10.21769/BioProtoc.3286.
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。