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Dec 2021

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Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, using an Escherichia coli host
使用大肠杆菌宿主的嗜热栖热菌重组醛脱氢酶的表达、纯化和体外酶活性测定   

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Abstract

Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDHTt from Thermus thermophilus, as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles.


In this protocol, we describe ALDHTt heterologous expression in E. coli, purification, and activity assay (based on Shortall et al., 2021). ALDHTt was first copurified as a contaminant during caa3-type cytochrome oxidase isolation from T. thermophilus. This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15–20 mg/L ALDHTt. For purification of the thermophilic his-tagged ALDHTt, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH).



Graphical abstract:



Flow chart outlining the steps in ALDHTt expression and purification, highlighting the approximate time required for each step.

Keywords: Aldehyde dehydrogenase (醛脱氢酶), Auto-induction media (自感应介质), Cell culture (细胞培养), Nickel affinity chromatography (镍亲和层析), Gel filtration chromatography (凝胶过滤层析), Heat treatment purification (热处理净化), UV-vis spectrophotometry (紫外-可见分光光度法), Enzymatic activity (酶活性)

Background

The aldehyde dehydrogenase ALDHTt was first identified and isolated as a contaminant, during cytochrome oxidase caa3-type native purification (Lyons et al., 2012) from Thermus thermophilus, which led to its further investigation. One of the common roles of ALDH family members in mammals is their inherent biosynthesis of retinoic acid assisted by cytochrome oxidases, which shows the possibility of such a role for this enzyme in T. thermophilus, due to the close relation between these two enzymes. Native purification of enzymes from T. thermophilus and other bacterial sources can be cumbersome, difficult, and lengthy, resulting in low protein yields (Soulimane, 2010; Robin et al., 2011). Therefore, recombinant protein production may be an attractive alternative route for the production of proteins for structural characterisation and functional analysis, allowing for high protein purity and yields. In 2018, the ALDHTt was first recombinantly expressed, purified, and its crystal structure determined (Hayes et al., 2018), revealing a novel C-terminal extension in the form of a tail, which contributes to active site regulation, thermostability and the oligomerization mode, aspects not before seen in the ALDH superfamily. The production protocol, employing a 48-h expression culture, heat treatment, immobilized metal affinity chromatography (IMAC) (Figure 1), and gel filtration chromatography, allows for yields of 15–20 mg of highly pure ALDHTt per litre of culture (Figure 2) (Shortall et al., 2021).


ALDHs are often not substrate specific, and can be characterised by their activity for the oxidation of aldehydes using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) to the corresponding carboxylic acid, and nicotinamide adenine dinucleotide (phosphate) hydrate (NAD(P)H). Like other dehydrogenase enzymes, their activity is monitored most commonly via UV-Vis spectrometry for the production of NAD(P)H at 340 nm (Figure 3). ALDHTt can oxidise a range of aldehydes, including aliphatic, aromatic, and cyclics, at elevated temperatures, with the highest catalytic activity achieved with hexanal at 50°C (Shortall et al., 2021).


Conditions detailed here allowed for the production of a pure, soluble, and active form of ALDHTt. The protocol described can also serve as a starting strategy to express and purify similar proteins.

Materials and Reagents

Note: All reagents were stored at room temperature, unless otherwise stated here.


  1. Ice

  2. MilliQ water


  1. Protein Expression

    1. 1.5 mL centrifuge tubes (Eppendorf, Sigma-Aldrich, catalog number: EP0030120086-1PAK)

    2. 50 mL sterile centrifuge tubes (Corning, Sigma-Aldrich, catalog number: CLS430290)

    3. 100 mm Petri dishes (Sigma-Aldrich, catalog number: P5731-500EA)

    4. Cell spreader, sterile (Sigma-Aldrich, catalog number: HS8171A-500EA)

    5. 2 L cell culture Erlenmeyer flasks

    6. 10 mL serological pipettes, sterile (Sigma-Aldrich, catalog number: CLS4488-50EA)

    7. 25 mL serological pipettes, sterile (Sigma-Aldrich, catalog number: CLS4251-200EA)

    8. Aluminium foil

    9. pET22b(+)-ALDHTt (constructed in the lab by Hayes et al. (2018)), store at -20°C

    10. BL21(DE3) chemically competent cells (prepared in the lab), store at -80°C

    11. LB agar (Fisher Scientific, catalog number: BP9724-500)

    12. LB broth (Sigma-Aldrich, catalog number: L3022)

    13. Ampicillin sodium salt (Fisher Scientific, catalog number: A0166), store at 4°C

    14. Tryptone (Fisher Scientific, catalog number: 1278-7099)

    15. Yeast extract (Fisher Scientific, catalog number: 10225203)

    16. Ammonium sulfate ((NH4)2SO4) (Sigma-Aldrich, catalog number: A4418)

    17. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662)

    18. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S9763)

    19. Glycerol (Fisher Scientific, catalog number: BP229-4)

    20. (D)-(+)-Glucose (Sigma-Aldrich, catalog number: G7528)

    21. α-Lactose monohydrate (Sigma-Aldrich, catalog number: L2643)

    22. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391)

    23. ZY Media (see Recipes)

    24. 20× NPS (see Recipes)

    25. 50× 5052 (see Recipes)

    26. 1 M MgSO4 (see Recipes)

    27. ZYP-5052 Auto-induction Media (see Recipes)


  2. Protein Purification

    1. 1 L Super-Speed Centrifuge Bottles with Sealing Closure, Nalgene (ThermoFisher, Fisher Scientific, catalog number: 3140-1006)

    2. 50 mL Oak Ridge High-Speed Polycarbonate Centrifuge Tubes w/Sealing Cap (ThermoFisher, Fisher Scientific, catalog number: 3138-0050PK)

    3. 10 mL serological pipettes, sterile (Sigma-Aldrich, catalog number: CLS4488-50EA)

    4. 50 mL sterile centrifuge tubes (Corning, Sigma-Aldrich, catalog number: CLS430290)

    5. 0.45 μm syringe filters, nylon (Fisher Scientific, catalog number: 15131499)

    6. 20 mL plastic syringes (Fisher Scientific, catalog number: 15889152)

    7. 500 mL glass beaker

    8. 4 L glass beaker

    9. Magnetic stir bars

    10. Dialysis clips

    11. Dialysis tubing, BiodesignTM Cellulose Dialysis Tubing Roll, 8000 Da MWCO (Fisher Scientific, catalog number: 12707486), store at 4°C

    12. Amicon Ultra-15 centrifugal filters, 50 kDa MWCO (Merck Millipore, Sigma-Aldrich, catalog number: UFC9050)

    13. 200 μL PCR tubes (Sigma-Aldrich, catalog number: BR781301)

    14. Liquid nitrogen (LN2)

    15. Lysozyme from chicken egg white (~ 70,000 U/mg) (Sigma-Aldrich, catalog number: 62971), store at 4°C

    16. Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25), store at -20°C

    17. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266)

    18. Trizma base (Sigma-Aldrich, catalog number: T6066)

    19. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 10053023)

    20. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148)

    21. Imidazole (Sigma-Aldrich, catalog number: I2399)

    22. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)

    23. Chelating Sepharose fast flow (Sigma-Aldrich, catalog number: GE17-0575-01), store at 4–30°C

    24. Ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)

    25. Nickel sulfate hexahydrate (NiSO4·6H2O) (Sigma-Aldrich, catalog number: 227676)

    26. Sodium acetate (C2H3NaO2) (Sigma-Aldrich, catalog number: S2889)

    27. Acetic acid (CH3COOH) (Sigma-Aldrich, catalog number: 695092)

    28. Lysis Buffer (see Recipes)

    29. Buffer A (see Recipes)

    30. Buffer B (see Recipes)

    31. Dialysis Buffer (see Recipes)

    32. Gel Filtration Buffer (see Recipes)


  1. ALDHTt Enzyme Assay

    1. Plastic cuvettes, Fisherbrand Macrocuvettes (Fisher Scientific, catalog number: FB55923)

    2. Hexanal (Sigma-Aldrich, catalog number: 115606)

    3. β-nicotinamide adenine dinucleotide sodium salt (NAD+) (Sigma-Aldrich, catalog number: N0632), store at -20°C

    4. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662)

    5. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786)

Equipment

  1. Biological safety cabinet

  2. Autoclave

  3. Lab balances

  4. Water bath

  5. Corning® 5 × 7 Inch Top PC-420D Stirring Hot Plate with Digital Displays, 120V/60Hz (Corning, catalog number: 6795-420D)

  6. Shaking incubator (Eppendorf, New Brunswick Scientific Inova 40, Sigma-Aldrich, catalog number: EPM1299-0094)

  7. -80°C freezer, New Brunswisk Scientific Ultra Low Temperature Freezer

  8. Large scale centrifuge (ThermoFisher Scientific Sorvall RC6+ Centrifuge, Fisher Scientific, catalog number: 12121680)

  9. ThermoFisher Scientific, Heraeus Megafuge 16R Centrifuge (ThermoFisher Scientific, Fisher Scientific, catalog number: 75004230)

  10. VWR Microstar12 microcentrifuge (VWR, catalog number: 521-1651)

  11. Probe sonicator (Bandelin Sonoplus HD 2200, catalog number: 2531)

  12. Peristaltic Pump P-1 (Cytivia Life Sciences, catalog number: 18111091)

  13. ÄKTAprime plus (Cytiva Life Sciences)

  14. XK16 chromatography column (Sigma-Aldrich, catalog number: GE28-9889-37)

  15. HiLoad 16/60 superdex 200 pg gel filtration column (Cytiva, Sigma-Aldrich, catalog number: GE28-9893-36)

  16. NanoDropTM ND-1000 (ThermoFisher Scientific, catalog number: ND-ONE-W)

  17. SDS-PAGE apparatus (Biorad Mini-PROTEAN Tetra Cell, catalog number: 1658005EDU)

  18. Cary 60 UV-vis spectrophotometer (Agilent) equipped with a temperature controller (TC-1 temperature controller) (Quantum Northwest, catalog number: TC 1-MAN-2.2)

  19. pH meter, Thermo Scientific Orion 2-star benchtop pH meter (Fisher Scientific, catalog number: Meter Kit 1111001)

Procedure

  1. Protein Expression

    1. Transformation: Transform the construct DNA pET-22b(+)-ALDHTt (Hayes et al., 2018) into E. coli BL21(DE3) chemically competent cells, via a standard heat shock in a water bath at 42°C for 30 s. Plate the resultant transformation on an agar plate supplemented with 100 μg/mL ampicillin, and grow at 37°C overnight.

    2. Preculture: Innoculate 10 mL of LB broth supplemented with 100 μg/mL ampicillin with one colony from the transformation plate, and incubate with agitation at 250 rpm and 37°C for 16 h.

    3. Main culture: Innoculate 1 L of ZYP-5052 auto-induction media (see Recipes below) supplemented with 50 μg/mL ampicillin with 10 mL of preculture (1% v/v), and incubate with agitation at 200 rpm and 25°C for 48 h.

      Note: The main culture for expression is split, so as to have no more than 500 mL in a 2- L shake flask, for aeration purposes.


  2. Protein Purification

    1. Transfer E. coli cells to 1 L centrifuge bottles, and harvest the cells via centrifugation at 6,000 × g and 4°C for 15 min.

      Note: Prior to cell collection, weigh the empty centrifuge bottles, to allow for determination of the cell mass after centrifuging.

    2. Discard the supernatant, and autoclave the waste.

    3. Weigh the cell pellet (typically ~35 g/L culture).

    4. Supplement the lysis buffer with 0.25 mg/mL lysozyme, 20 μg/mL DNase I, and 200 mM MgCl2. Resuspend the pellet in lysis buffer (see Recipes below), adding 5 mL of lysis buffer for every 1 g of cells present.

      Note: Resuspension of cells is carried out on ice using a 10-mL serological pipette for drawing the buffer-cell suspension up and down. If carried out at room temperature, this should be performed in a timely manner, as native cell proteases may degrade the desired protein.

    5. Store the cell lysate in aliquots of 50 mL in Falcon tubes at -80°C overnight.

      Note: Storage of cell lysate at -80°C overnight can be extended for up to 1 week.

    6. Thaw the cell lysate in a water bath at 37°C.

    7. Sonicate the cells using a probe sonicator with the power set between 60–70% (ultrasonic nominal output maximum of 200 W). Sonicate for 30 s, rest for 30 s, and repeat this process three times.

      Note: Sonication was carried out in a cold room at 4°C, with the cell lysate solution surrounded by ice.

    8. Heat the cell lysate in a water bath to 65°C for 15 min. Omission of this step results in an approximate 40% decrease in protein yield.

    9. Collect the cell debris via centrifugation at 34,000 × g and 4°C for 30 min. Remove the supernatant that contains the ALDHTt, and store on ice. Autoclave the collected cells prior to disposal.

      Note: Following the 30-min centrifugation for collection of the soluble fraction containing ALDHTt, the sample should be collected immediately after the centrifuge stops, to ensure the cells don’t re-enter solution.

    10. Filter the collected soluble fraction through a 0.45 μm nylon syringe filter, and store in a 50 mL Falcon tube on ice. Keep 1 mL of sample for SDS-PAGE and Western blot analysis.

    11. Pack an XK 16/20 column with approximately 10 mL of chelating Sepharose fast flow resin, and pre-equilibrate as follows. Using a peristaltic pump, deliver the following quantities of buffer to the column, at a rate of 1–3 mL/min: 0.5 column volume (CV) of 0.2 M EDTA, 0.5 M NaCl, pH 7, 2 CV of 0.5 M NaCl, 2 CV of MilliQ water, 0.2 CV of 0.2 M NiSO4, 5 CV of MilliQ water, 5 CV of 20 mM sodium acetate, 0.5 M NaCl pH 4, and finally 2 CV of buffer A (see Recipes below).

    12. Load the entire filtered soluble fraction (approximately 80–100 mL from 1 L culture) onto the Ni-affinity column. Keep the solution on ice during loading. Collect the flow through for SDS-PAGE and Western blot analysis.

    13. Connect the column to the Akta Prime system, and start the IMAC purification.

    14. Wash the column with buffer A at 3 mL/min, until a stable (close to zero) Abs280nm is achieved.

    15. Elute proteins at 3 mL/min, via an imidazole step gradient of 50, 100, 200, and 500 mM (Figure 1), using a combination of buffer A and buffer B (see Recipes below). Only increase the concentration of buffer B, and thus imidazole, when the Abs280nm obtained from the previous concentration is stable. Collect a fraction from each elution for SDS-PAGE and Western blot.

      Note: ALDHTt should elute from Ni-affinity chromatography in the 200 mM imidazole fraction.



      Figure 1. Elution profile of ALDHTt via Ni-affinity chromatography.

      Chromatogram displaying purification of ALDHTt via Ni-affinity chromatography using a step gradient of imidazole from 50–500 mM. E. coli host proteins are eluted from 10–50 mM imidazole in the first two peaks, while ALDHTt is eluted at 200 mM imidazole in the third peak.



      Figure 2. SDS-PAGE of ALDHTt expression and purification samples.

      Lane 1: PageRuler Pre-stained protein ladder (ThermoFisher Scientific), lane 2: E. coli BL21(DE3) cell lysate expressing ALDHTt, lane 3: Ni-affinity chromatography 200 mM imidazole elution, lane 4: purified ALDHTt following gel filtration chromatography, lane 5: Ni-affinity chromatography flow through (adapted from Shortall et al., 2021).


    16. Take the fractions containing the ALDHTt (typically 30–40 mL), and dialyse against 4 L of dialysis buffer (see Recipes below). Dialyse with gentle magnetic stirring at 4°C overnight.

    17. Concentrate the protein sample using Amicon Ultra-15 centrifugal filters, 50 kDa MWCO (Merck Millipore), at 3,500 × g using 5 min spins, until a volume of approximately 1 mL (containing approximately 15–20 mg/mL) is obtained.

    18. Gel filtration chromatography: Pre-equilibrate the HiLoad 16/60 Superdex 200 pg column with 2 CV (240 mL) of gel filtration buffer (see Recipes below) at 1 mL/min. Load approximately 1 mL of concentrated ALDHTt protein sample onto the column using the injection loop on the Akta Prime system, and run at 1 mL/min for 120 min, with collection of the protein peak.

      Note: ALDHTt should begin to elute from gel filtration chromatography at approximately 65 mL. There should only be one peak on the gel filtration chromatogram. A slight shoulder at the start of the peak (on the left) can occur, but should not be collected.

    19. Pool the fractions from gel filtration chromatography (approximately 20 mL), and concentrate to 25–30 mg/mL (or as desired) using Amicon Ultra-15 centrifugal filters, 50 kDa MWCO (Merck Millipore), at 3,500 × g using 5 min spins, until the desired concentration is obtained.

    20. Monitor protein concentration by UV absorbance at 280 nm on a NanoDropTM 1000 spectrophotometer, using a sequence-derived extinction coefficient of 1671 M-1 cm-1. Use gel filtration buffer as the blank.

    21. Prepare 30-μL aliquots, snap freeze in liquid nitrogen (LN2), and store at -80°C until further use. If the protein is to be used immediately, aliquot as desired, and store on ice until further use.


  3. ALDHTt Enzyme Assay

    1. Assay the ALDHTt, using hexanal as the substrate (2 mM) and NAD+ as the cofactor (2 mM), in 10 mM potassium phosphate pH 8.

      Note: Hexanal is only slightly soluble in water, so stock solutions at low concentrations should be prepared. Solubility in water is ~6 g/L (60 mM). Shelf life of the hexanal solution prepared in buffer is one week.

    2. Program the spectrophotometer equipped with a temperature controller to analyse at 340 nm and 50°C for 2 min. Add a pre-equilibration step of 1 min, to ensure all solutions are kept at 50°C. A short analysis time of 2 min is utilised to avoid loss of volatile aldehyde substrates due to evaporation.

    3. Heat solutions of 5 mM hexanal, 10 mM NAD+, and 10 mM potassium phosphate pH 8 to 50°C, in a water bath.

      Note: The enzyme activity of ALDHTt can be analysed at 20–50°C. However, with decreased temperature a decrease in enzymatic activity is observed.

    4. Thaw a 30-μL aliquot of ALDHTt on ice.

    5. Dilute the ALDHTt to obtain a concentration of 0.38 mg/mL using gel filtration buffer, and centrifuge at 4,050 × g for 5 min.

      Note: Diluting the ALDHTt to a concentration of 0.38 mg/mL for the enzyme assay should be carried out using gel filtration buffer. For example, a 1:70 dilution of ALDHTt at a stock concentration of 26.65 mg/mL can be performed, by adding 10 μL of ALDHTt to 690 μL of gel filtration buffer.

    6. To a plastic cuvette, add 360 μL of 10 mM NAD+, 680 μL of 10 mM potassium phosphate pH 8, and 40 μL of ALDHTt at 0.38 mg/mL. Insert the cuvette into the spectrophotometer, to undergo equilibration at 50°C for 1 min.

    7. Add 720 μL of 5 mM hexanal to the cuvette and mix gently. Monitor the absorbance at 340 nm for 2 min, for the production of NADH.

      Note: Enzymatic assaying of ALDHTt should result in an increase in absorbance at 340 nm (a positive slope) when monitored at 50°C (Figure 3).

    8. Calculate enzyme activity employing Beer-Lambert’s Law, using the 6,220 M-1 cm-1 extinction coefficient of NADH at 340 nm (Equation 1).



      Note: One enzyme unit is equal to the production of 1 µmol/min NADH. l is equal to the path length of the cuvette, in this case l = 1 cm. Vol is equal to the volume of the cuvette, in this case vol = 1.8 mL.



    Figure 3. Principle for ALDHTt assaying using hexanal and NAD+ with activity monitored spectrophotometrically via the appearance of NADH. An example of the cuvette used and results obtained are demonstrated.

Data analysis

The original research article for the above expression, purification, and enzyme activity analysis was published in Shortall et al. (2021), https://doi.org/10.3390/cells10123535.

Recipes

Note: All solutions were prepared using MilliQ water (18.2 MΩ cm).


  1. ZY Media

    10% typtone

    5% yeast extract

  2. 20× NPS (NPS = 100 mM PO4, 25 mM SO4, 50 mM NH4, 100 mM Na, 50 mM K)

    0.5 M (NH4)2SO4

    1 M KH2PO4

    1 M Na2HPO4

  3. 50× 5052 (5052 = 0.5% glycerol, 0.05% glucose, 0.2% α-lactose)

    25% glycerol

    2.5% glucose

    10% α-lactose
    Note: Preparation of 50× 5052 can be assisted by gentle heating with magnetic stirring. 50× 5052 can be difficult to dissolve, and should not be autoclaved until all components are dissolved. If the solution is visibly displaying a brown tinge after autoclaving, it should be re-prepared.

  1. 1 M MgSO4

  2. ZYP-5052 Auto-induction Media

    ~928 mL of ZY media

    1 mL of 1 M MgSO4

    20 mL of 50× 5052

    50 mL of 20× NPS

    50 μg/mL ampicillin

    Note: Solutions for ZYP-5052 auto-induction media are prepared and autoclaved separately. These are combined to make the media on the day of the expression culture.

  3. Lysis Buffer

    20 mM Tris-HCl pH 7.5

    5 mM β-mercaptoethanol

    10 mM imidazole

    500 mM NaCl

  4. Buffer A

    20 mM Tris-HCl pH 7.5

    5 mM β-mercaptoethanol

    10 mM imidazole

    200 mM NaCl

  5. Buffer B

    20 mM Tris-HCl pH 7.5

    5 mM β-mercaptoethanol

    1 M imidazole

    200 mM NaCl

  6. Dialysis Buffer

    50 mM Tris-HCl pH 7.5

    5 mM β-mercaptoethanol

    250 mM NaCl

  7. Gel Filtration Buffer

    50 mM Tris-HCl pH 7.5

    5 mM β-mercaptoethanol

    150 mM NaCl

Acknowledgments

This protocol is an extension of that described in Shortall et al. (2021) and adapted from Hayes et al. (2018). This research was funded by European Union’s Horizon 2020 Research and Innovation programme, Oyster (Open characterisation and modelling environment to drive innovation in advanced nano-architectured and bio-inspired hard/soft interfaces) under grant agreement No. 760827. Funding is also acknowledged from the Department of Chemical Sciences, University of Limerick and The Higher Education Authority, Ireland.

Competing interests

There are no conflicts of interest or competing interests.

References

  1. Hayes, K., Noor, M., Djeghader, A., Armshaw, P., Pembroke, T., Tofail, S. and Soulimane, T. (2018). The quaternary structure of Thermus thermophilus aldehyde dehydrogenase is stabilized by an evolutionary distinct C-terminal arm extension. Sci Rep 8(1): 1-14.
  2. Lyons, J. A., Aragão, D., Slattery, O., Pisliakov, A. V., Soulimane, T. and Caffrey, M. (2012). Structural insights into electron transfer in caa 3-type cytochrome oxidase. Nature 487(7408): 514-518.
  3. Robin, S., Arese, M., Forte, E., Sarti, P., Giuffre, A. and Soulimane, T. (2011). A sulfite respiration pathway from Thermus thermophilus and the key role of newly identified cytochrome c550. J Bacteriol 193(15): 3988-3997.
  4. Shortall, K., Durack, E., Magner, E. and Soulimane, T. (2021). Study of ALDH from Thermus thermophilus-Expression, Purification and Characterisation of the Non-Substrate Specific, Thermophilic Enzyme Displaying Both Dehydrogenase and Esterase Activity. Cells 10(12): 3535.
  5. Soulimane, T. (2010). Thermus thermophilus encodes an archaeal-like fructose-1,6-bisphosphatase: purification of native and recombinant protein for structural studies. Protein Expr Purif 74(2): 175-180.

简介

[摘要]基于前人的深入表征,醛脱氢酶(ALDH)是一种酶的超家族,在结构和功能上都存在于生命的各个领域。它们使用辅因子烟酰胺腺嘌呤二核苷酸(磷酸盐)(NAD(P) + )催化醛氧化成羧酸,并且通常不是底物特异性的,而是具有广泛的相关生物学功能,包括解毒和生物合成.我们研究了来自嗜热栖热菌的ALDH Tt的结构,并对其进行了生化表征。这允许深入了解其潜在的底物和生物学作用。
在本协议中,我们描述了ALDH Tt在大肠杆菌中的异源表达、纯化和活性测定(基于 Shortall等人,2021)。 ALDH Tt在从嗜热嗜热杆菌中分离出caa 3型细胞色素氧化酶时首次作为污染物被共纯化。该重组生产系统用于野生型和突变体的结构和生化分析,证明是有效的,产生大约 15 – 20 mg/L ALDH Tt 。为了纯化嗜热的带his标签的ALDH Tt ,使用热处理、固定化金属亲和层析(IMAC)和凝胶过滤层析。通过紫外-可见分光光度法进行酶活性测定,监测还原的烟酰胺腺嘌呤二核苷酸(NADH)的产生。

图形概要:

流程图概述了ALDH Tt表达和纯化的步骤,突出了每个步骤所需的大致时间。

[背景] 醛脱氢酶ALDH Tt在嗜热栖热菌的细胞色素氧化酶 caa 3型天然纯化(Lyons et al. , 2012)期间首次被鉴定和分离为污染物,这导致了进一步的研究。 ALDH 家族成员在哺乳动物中的共同作用之一是它们在细胞色素氧化酶的辅助下固有的视黄酸生物合成,这表明这种酶在嗜热菌中发挥这种作用的可能性,因为这两种酶之间的密切关系。从嗜热链球菌和其他细菌来源中天然纯化酶可能很麻烦、困难且耗时,导致蛋白质产量低(Soulimane,2010;Robin等人,2011) 。因此,重组蛋白质生产可能是用于生产用于结构表征和功能分析的蛋白质的有吸引力的替代途径,从而实现高蛋白质纯度和产量。 2018 年, ALDH Tt首次被重组表达、纯化,并确定了其晶体结构(Hayes等,2018) ,揭示了一种新的尾部形式的 C 末端延伸,有助于活性位点调节、热稳定性和低聚模式,在 ALDH 超家族中从未见过的方面。该生产方案采用 48 小时表达培养、热处理、固定化金属亲和层析 (IMAC)(图 1)和凝胶过滤层析,每升培养物可产生 15 – 20 mg高纯度ALDH Tt (图 2) (Shortall等人,2021 年) 。
ALDHs 通常不是底物特异性的,其特征在于它们使用辅因子烟酰胺腺嘌呤二核苷酸(磷酸盐)(NAD(P) + )将醛氧化为相应的羧酸和烟酰胺腺嘌呤二核苷酸(磷酸盐)水合物的活性。 NAD(P)H)。与其他脱氢酶一样,它们的活性最常通过 UV-Vis 光谱法监测,以在 340 nm 处产生 NAD(P)H(图 3)。 ALDH Tt可以在高温下氧化一系列醛,包括脂肪族、芳香族和环状醛,在 50°C 下使用己醛实现最高催化活性(Shortall等人,2021 年) 。
此处详述的条件允许生产纯的、可溶的和活性形式的ALDH Tt 。所描述的协议也可以作为表达和纯化类似蛋白质的起始策略。

关键字:醛脱氢酶, 自感应介质, 细胞培养, 镍亲和层析, 凝胶过滤层析, 热处理净化, 紫外-可见分光光度法, 酶活性

材料和试剂
注意:除非另有说明,否则所有试剂均在室温下保存。

MilliQ水
蛋白质表达
1.5 mL离心管(Eppendorf,Sigma-Aldrich,目录号:EP0030120086-1PAK)
50 mL无菌离心管(Corning, Sigma-Aldrich,目录号:CLS430290)
100毫米培养皿(Sigma-Aldrich,目录号:P5731-500EA)
细胞吊具,无菌(Sigma-Aldrich,目录号:HS8171A-500EA)
2 L 细胞培养锥形瓶
10 mL血清移液管,无菌(Sigma-Aldrich,目录号:CLS4488-50EA)
25 mL血清移液管,无菌(Sigma-Aldrich,目录号:CLS4251-200EA)
铝箔_
pET22 b( +)- ALDH Tt (由 Hayes等人在实验室构建(2018 年)),储存于 -20°C
BL21(DE3) 化学感受态细胞(实验室制备),-80°C 储存
LB琼脂(Fisher Scientific,目录号:BP9724-500)
LB肉汤(Sigma-Aldrich,目录号:L3022)
氨苄青霉素钠盐(Fisher Scientific,目录号: A0166),储存在 4°C
胰蛋白胨(Fisher Scientific,目录号:1278-7099)
酵母提取物(Fisher Scientific,目录号:10225203)
硫酸铵((NH 4 ) 2 SO 4 )(Sigma-Aldrich,目录号:A4418)
磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P0662)
磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:S9763)
甘油(Fisher Scientific,目录号:BP229-4)
(D)-(+)-葡萄糖(Sigma-Aldrich,目录号:G7528)
α-乳糖一水合物(Sigma-Aldrich,目录号:L2643)
七水合硫酸镁(MgSO 4 · 7H 2 O)(Sigma-Aldrich,目录号:230391)
ZY 媒体(见食谱)
20 × NPS(见食谱)
50 × 5052(见配方)
1 M MgSO 4 (见配方)
ZYP-5052 自动感应培养基(见配方)


蛋白质纯化
1 L 带密封盖的超高速离心瓶,Nalgene( ThermoFisher ,Fisher Scientific,目录号:3140-1006)
50 mL Oak Ridge 高速聚碳酸酯离心管,带密封盖( ThermoFisher ,Fisher Scientific,目录号:3138-0050PK)
10 mL血清移液管,无菌(Sigma-Aldrich,目录号:CLS4488-50EA)
50 mL无菌离心管(Corning,Sigma-Aldrich,目录号:CLS430290)
0.45 μm注射器过滤器,尼龙(Fisher Scientific,目录号:15131499)
20 mL 塑料注射器(Fisher Scientific,目录号:15889152)
500 毫升玻璃烧杯
4升玻璃烧杯
磁力搅拌棒
透析夹
透析管, Biodesign TM纤维素透析管卷,8000 Da MWCO(Fisher Scientific,目录号:12707486),在 4°C 下储存
Amicon Ultra-15 离心过滤器,50 kDa MWCO(Merck Millipore,Sigma-Aldrich,目录号:UFC9050)
200 μL PCR管(Sigma-Aldrich,目录号:BR781301)
液氮 (LN 2 )
来自鸡蛋清的溶菌酶(~ 70,000 U/mg)(Sigma-Aldrich,目录号:62971),储存于 4°C
来自牛胰腺的脱氧核糖核酸酶I(Sigma-Aldrich,目录号:DN25),储存在-20°C
氯化镁(MgCl 2 )(Sigma-Aldrich,目录号:M8266)
Trizma碱(Sigma-Aldrich,目录号:T6066)
盐酸(HCl)(Fisher Scientific,目录号:10053023)
β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
咪唑(Sigma-Aldrich,目录号:I2399)
氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
螯合琼脂糖快速流动(Sigma-Aldrich,目录号:GE17-0575-01),储存在 4 – 30°C
乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
硫酸镍六水合物(NiSO 4 · 6H 2 O)(Sigma-Aldrich,目录号:227676)
乙酸钠(C 2 H 3 NaO 2 )(Sigma-Aldrich,目录号:S2889)
乙酸(CH 3 COOH)(Sigma-Aldrich,目录号:695092)
裂解缓冲液(见配方)
缓冲区 A(见配方)
缓冲液 B(见配方)
透析缓冲液(见配方)
凝胶过滤缓冲液(参见配方)


ALDH Tt酶检测
塑料比色皿, Fisherbrand Macrocuvettes (Fisher Scientific,目录号:FB55923)
己醛(Sigma-Aldrich,目录号:115606)
β-烟酰胺腺嘌呤二核苷酸钠盐(NAD + )(Sigma-Aldrich,目录号:N0632),储存于-20°C
磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P0662)
磷酸氢二钾(K 2 HPO 4 )(Sigma-Aldrich,目录号:P3786)


设备


生物安全柜
高压釜
实验室天平
水浴
Corning ® 5 × 7 英寸顶部 PC-420D 带数字显示屏的搅拌热板,120V/60Hz(Corning,目录号:6795-420D)
摇动培养箱(Eppendorf,New Brunswick Scientific Inova 40,Sigma-Aldrich,目录号:EPM1299-0094)
-80°C 冰箱,New Brunswisk Scientific 超低温冰箱
大型离心机(ThermoFisher Scientific Sorvall RC6+ Centrifuge,Fisher Scientific,目录号:12121680)
ThermoFisher Scientific,Heraeus Megafuge 16R 离心机(ThermoFisher Scientific,Fisher Scientific,目录号:75004230)
VWR Microstar12 微型离心机(VWR,目录号:521-1651)
探头超声仪(Bandelin Sonoplus HD 2200,目录号:2531)
蠕动泵 P-1(Cytivia Life Sciences,目录号:18111091)
ÄKTAprime plus(Cytiva 生命科学)
XK16色谱柱(Sigma-Aldrich,目录号:GE28-9889-37)
HiLoad 16/60 superdex 200 pg凝胶过滤柱(Cytiva,Sigma-Aldrich,目录号:GE28-9893-36)
NanoDrop TM ND-1000(ThermoFisher Scientific,目录号:ND-ONE-W)
SDS-PAGE 仪器(Biorad Mini-PROTEAN Tetra Cell,目录号:1658005EDU)
配备温度控制器(TC-1 温度控制器)(Quantum Northwest,目录号:TC 1-MAN-2.2)的 Cary 60 紫外可见分光光度计(Agilent)
pH计,Thermo Scientific Orion 2星台式pH计(Fisher Scientific,目录号:Meter Kit 1111001)


程序


蛋白质表达
转化:转化构建体 DNA pET-22b(+)- ALDH Tt (Hayes et al. , 2018)进入大肠杆菌BL21(DE3) 化学感受态细胞,通过标准热激在 42°C 的水浴中持续 30 秒。将产生的转化铺板在补充有 100 μg /mL 氨苄青霉素的琼脂板上,并在 37°C 下生长过夜。
预培养:用转化板中的一个菌落接种10 mL 补充有 100 μg /mL 氨苄青霉素的 LB 肉汤,并在 250 rpm 和 37°C 下搅拌孵育 16 h。
主要培养物:接种 1 L ZYP -5052 自诱导培养基(参见以下配方),添加 50 μg /mL 氨苄青霉素和 10 mL 预培养物(1% v/v),并在 200 rpm 和 25°C 下搅拌孵育48 小时。
笔记: 用于表达的主要培养物是分开的,以便在 2-L 摇瓶中不超过 500 mL,用于通气目的。


蛋白质纯化
将大肠杆菌细胞转移到 1 L 离心瓶中,并在 6,000 × g和4°C 下离心 15 分钟以收获细胞。
注意:在细胞收集之前,称量空离心瓶,以便在离心后确定细胞质量。
丢弃上清液,并对废物进行高压灭菌。
称量细胞颗粒(通常为 ±35 g/L 培养物)。
μg /mL DNase I 和 200 mM MgCl 2补充裂解缓冲液。将颗粒重新悬浮在裂解缓冲液中(参见下面的配方),每 1 g 细胞加入 5 mL 裂解缓冲液。
注意:使用 10 mL 血清移液管在冰上进行细胞重悬,以上下吸取缓冲细胞悬液。如果在室温下进行,则应及时进行,因为天然细胞蛋白酶可能会降解所需的蛋白质。
将细胞裂解物以 50 mL 的等分试样储存在 -80°C 的 Falcon 管中过夜。
注意:细胞裂解液在 -80°C 下的过夜储存可延长至 1 周。
在 37°C 的水浴中解冻细胞裂解物。
功率设置在60-70 %之间的探头声波器对细胞进行声波处理(超声波标称输出最大为 200 W)。声波处理 30 s,休息 30 s,并重复此过程 3 次。
注意:超声处理是在 4°C 的冷室中进行的,细胞裂解液被冰包围。
在水浴中将细胞裂解物加热至 65°C 15 分钟。省略此步骤会导致蛋白质产量降低约 40%。
× g和 4°C 下离心 30 分钟收集细胞碎片。除去含有ALDH Tt的上清液,并储存在冰上。在处置前对收集的细胞进行高压灭菌。
注意:在离心 30 分钟以收集含有ALDH Tt的可溶性部分后,应在离心机停止后立即收集样品,以确保细胞不会重新进入溶液。
通过 0.45 μm尼龙注射器过滤器过滤收集的可溶性部分,并储存在冰上的 50 mL Falcon 管中。保留 1 mL 的样品用于 SDS-PAGE 和西方印迹分析。
用大约 10 mL 的螯合 Sepharose 快速流动树脂填充 XK 16/20 色谱柱,并按如下方式进行预平衡。使用蠕动泵,以 1 – 3 mL/min的速率将以下数量的缓冲液输送到柱中:0.5 柱体积 (CV) 的 0.2 M EDTA、0.5 M NaCl、pH 7、2 CV 的 0.5 M NaCl 、2 CV MilliQ水、0.2 CV 0.2 M NiSO 4 、5 CV MilliQ水、5 CV 20 mM 乙酸钠、0.5 M NaCl pH 4,最后是 2 CV 缓冲液 A(参见下面的配方)。
将整个过滤的可溶性部分(从 1 L 培养物中大约 80-100 mL)加载到 Ni 亲和柱上。在装载过程中将溶液保持在冰上。收集用于 SDS-PAGE 和西方印迹分析的流量。
将色谱柱连接到Akta Prime 系统,并开始 IMAC 纯化。
以 3 mL/min 的速度用缓冲液 A 清洗色谱柱,直至达到稳定(接近于零)的 Abs 280nm 。
使用缓冲液 A 和缓冲液 B 的组合,通过 50、100、200 和 500 mM 的咪唑阶梯梯度(图 1)以 3 mL/min 的速度洗脱蛋白质(参见下面的食谱)。仅当从先前浓度获得的 Abs 280nm稳定时,才增加缓冲液 B 的浓度,从而增加咪唑。从每个洗脱中收集一部分用于 SDS-PAGE 和西方印迹。
注意: ALDH Tt应从 200 mM 咪唑馏分中的镍亲和色谱中洗脱。


 
图 1. ALDH Tt通过 Ni 亲和色谱的洗脱曲线。 
色谱图显示使用 50–500 mM的咪唑阶梯梯度通过 Ni 亲和色谱纯化ALDH Tt 。 大肠杆菌宿主蛋白在前两个峰中从 10-50 mM 咪唑中洗脱,而ALDH Tt在第三个峰中在 200 mM 咪唑中洗脱。


 
图 2. ALDH Tt表达和纯化样品的 SDS-PAGE。
泳道 1: PageRuler预染色蛋白阶梯 ( ThermoFisher Scientific),泳道 2:表达ALDH Tt的大肠杆菌BL21(DE3) 细胞裂解物,泳道 3:Ni 亲和层析 200 mM 咪唑洗脱,泳道 4:纯化的ALDH Tt 在凝胶过滤色谱之后,泳道 5:Ni 亲和色谱流过(改编自 Shortall等人,2021)。


取含有ALDH Tt (通常为 30 – 40 mL)的馏分,并用4 L 的透析缓冲液进行透析(参见下面的食谱)。在 4°C 下用温和的磁力搅拌透析过夜。
Amicon Ultra-15 离心过滤器、50 kDa MWCO (Merck Millipore) 以 3,500 × g浓缩蛋白质样品 使用 5 分钟旋转,直到获得大约 1 mL 的体积(包含大约 15 – 20 mg/mL)。
凝胶过滤色谱:以 1 mL/min 的速度预平衡HiLoad 16/60 Superdex 200 pg柱与 2 CV (240 mL) 凝胶过滤缓冲液(参见下面的配方)。使用Akta Prime 系统上的注射回路将大约 1 mL 的浓缩ALDH Tt蛋白质样品加载到色谱柱上,并以 1 mL/min 的速度运行 120 分钟,并收集蛋白质峰。
注意: ALDH Tt应该在大约 65 mL时开始从凝胶过滤色谱中洗脱。凝胶过滤色谱图上应该只有一个峰。峰开始处(左侧)可能会出现轻微的肩部,但不应收集。
将凝胶过滤色谱(约 20 mL)的级分合并,并使用Amicon Ultra-15 离心过滤器、50 kDa MWCO(Merck Millipore)浓缩至 25–30 mg/mL(或根据需要),在 3,500 × g下使用 5 分钟旋转,直到获得所需的浓度。
NanoDrop TM 1000 分光光度计上,使用 1671 M -1 cm -1的序列衍生消光系数,通过280 nm 处的紫外吸光度监测蛋白质浓度。使用凝胶过滤缓冲液作为空白。
准备 30-μL 等分试样,在液氮 (LN 2 ) 中快速冷冻,并储存在 -80°C 直至进一步使用。如果要立即使用蛋白质,则根据需要等分,并储存在冰上直至进一步使用。


ALDH Tt酶检测
在 10 mM 磷酸钾 pH 8 中,使用己醛作为底物 (2 mM) 和 NAD +作为辅因子 (2 mM)测定ALDH Tt 。
注意:己醛仅微溶于水,因此应准备低浓度的储备溶液。在水中的溶解度约为 6 g/L (60 mM)。在缓冲液中制备的己醛溶液的保质期为一周。
对配备温度控制器的分光光度计进行编程,在 340 nm 和 50°C 下分析 2 分钟。添加 1 分钟的预平衡步骤,以确保所有溶液保持在 50°C。使用 2 分钟的短分析时间来避免挥发性醛底物因蒸发而损失。
加热 5 mM 己醛、10 mM NAD +和 10 mM 磷酸钾 pH 8 至 50°C 的溶液。
ALDH Tt的酶活性可以在 20–50°C 下进行分析。然而,随着温度降低,观察到酶活性降低。
在冰上解冻 30 μL 的ALDH Tt等分试样。
稀释ALDH Tt以获得 0.38 mg/mL 的浓度,并以 4,050 × g离心5 分钟。
注意:应使用凝胶过滤缓冲液将ALDH Tt稀释至 0.38 mg/mL 的浓度进行酶测定。例如,通过将 10 μL的ALDH Tt添加到 690 μL的凝胶过滤缓冲液中,可以在 26.65 mg/mL 的储备浓度下进行1:70 的ALDH Tt稀释。
在塑料比色皿中,加入 360 μL的 10 mM NAD + 、680 μL的 10 mM 磷酸钾 pH 8 和 40 μL的ALDH Tt ,浓度为 0.38 mg/ mL。将比色皿插入分光光度计中,在 50°C 下平衡 1 分钟。
加入 720 μL的 5 mM 己醛并轻轻混合。监测 340 nm 的吸光度 2 分钟,以产生 NADH。
注: ALDH Tt的酶促测定 在 50°C 下监测时,应导致 340 nm 处的吸光度增加(正斜率)(图 3)。
使用比尔-朗伯定律计算酶活性,使用 NADH 在 340 nm 处的 6,220 M -1 cm -1消光系数(方程式 1)。


"                                 U " 〖"mg" 〗^"-1"  "= "  ("slope × " 〖"10" 〗^"6"  " × vol" )/("6220 " "M" ^"-1"  〖"cm" 〗^"-1 "  "× l × mg of " 〖"ALDH" 〗_"Tt"  )                     " Equation 1" 


注意:一个酶单位等于 1 µmol/min NADH 的产量。 l 等于比色皿的路径长度,在这种情况下 l = 1 cm。 Vol 等于比色皿的体积,在这种情况下 vol = 1.8 mL。 
 
+进行ALDH Tt测定的原理,通过 NADH 的出现通过分光光度法监测活性。展示了使用的比色皿示例和获得的结果。


数据分析


上述表达、纯化和酶活性分析的原始研究文章发表于 Shortall等人。 (2021 年), https://doi.org/10.3390/cells10123535 。


食谱


注意:所有溶液均使用MilliQ水 (18.2 MΩ cm) 制备。


中源传媒
10%色度 
5% 酵母提取物
20 × NPS(NPS= 100 mM PO 4 、25 mM SO 4 、50 mM NH 4 、100 mM Na、50 mM K)
0.5 M (NH 4 ) 2 SO 4
1 M KH 2 PO 4
1 M Na 2 HPO 4
50 × 5052(5052 = 0.5% 甘油、0.05% 葡萄糖、0.2% α-乳糖)
25% 甘油
2.5% 葡萄糖
10% α-乳糖
注意:50 × 5052 的制备可以通过磁力搅拌温和加热来辅助。 50 × 5052 可能难以溶解,在所有组分溶解之前不应高压灭菌。如果高压灭菌后溶液明显呈褐色,则应重新制备。
1M MgSO 4
ZYP-5052 自感应介质
~928 mL ZY 培养基
1 mL 1 M MgSO 4
20 毫升 50 × 5052
50 mL 的 20 × NPS
50微克/毫升氨苄青霉素
注意:ZYP-5052 自感应培养基的溶液单独制备和高压灭菌。这些结合起来,使媒体在当天表达文化。
裂解缓冲液
20 mM Tris-HCl pH 7.5
5 mM β-巯基乙醇
10 毫米咪唑
500 毫米氯化钠
缓冲器 A
20 mM Tris-HCl pH 7.5
5 mM β-巯基乙醇
10 毫米咪唑
200 毫米氯化钠
缓冲器 B
20 mM Tris-HCl pH 7.5
5 mM β -巯基乙醇
1M咪唑
200 毫米氯化钠
透析缓冲液
50 mM Tris-HCl pH 7.5
5 mM β-巯基乙醇 
250 毫米氯化钠
凝胶过滤缓冲液
50 mM Tris-HCl pH 7.5
5 mM β-巯基乙醇
150 毫米氯化钠


致谢


该协议是 Shortall等人描述的协议的扩展。 (2021) 并改编自 Hayes等人。 (2018 年)。这项研究由欧盟的 Horizon 2020 研究和创新计划Oyster(开放表征和建模环境,以推动先进纳米架构和仿生硬/软接口的创新)根据第 760827 号赠款协议资助。资金也来自利默里克大学化学科学系和爱尔兰高等教育局。


利益争夺


不存在利益冲突或竞争利益。 


参考


Hayes, K., Noor, M., Djeghader , A., Armshaw , P., Pembroke, T., Tofail , S. 和Soulimane , T. (2018)。嗜热栖热菌醛脱氢酶的四级结构通过进化上独特的 C 末端臂延伸来稳定。 科学代表8(1):1-14。
Lyons, JA, Aragão , D., Slattery, O., Pisliakov , AV, Soulimane , T. 和 Caffrey, M. (2012)。 caa 3 型细胞色素氧化酶中电子转移的结构见解。 自然487(7408):514-518。
Robin, S.、Arese, M.、Forte, E.、 Sarti , P.、Giuffre, A. 和Soulimane , T. (2011)。嗜热栖热菌的亚硫酸盐呼吸途径和新发现的细胞色素 c 550的关键作用。 细菌杂志193(15):3988-3997 。
Shortall, K., Durack, E., Magner, E. 和Soulimane , T. (2021)。来自嗜热栖热菌的 ALDH 的研究 -表现出脱氢酶和酯酶活性的非底物特异性嗜热酶的表达、纯化和表征。 单元格10(12):3535。
苏里曼,T. (2010)。嗜热栖热菌编码一种类似古菌的果糖-1,6-双磷酸酶:纯化天然和重组蛋白用于结构研究。 蛋白质表达纯化74(2):175-180 。


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引用:Shortall, K., Magner, E. and Soulimane, T. (2022). Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, using an Escherichia coli host. Bio-protocol 12(9): e4401. DOI: 10.21769/BioProtoc.4401.
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