Protocol for the purification, analysis, and handling of acyl carrier proteins from type I fatty acid and polyketide synthases

Summary

Acyl carrier proteins (ACPs) are key domains in fatty acid and polyketide synthases (PKSs), shuttling intermediates to catalytic partners. Here, we present a protocol for purifying and analyzing ACPs from fatty acid synthases (FASs) and PKSs, using four model ACPs from Mus musculus, Saccharopolyspora erythraea, Streptomyces venezuelae, and Streptomyces hygroscopicus. We describe steps for recombinant ACP production in E. coli; purification via chromatography or precipitation; ACP modification (holo/acyl forms); and analysis using urea PAGE, high-performance liquid chromatography (HPLC), and NMR spectroscopy.

For complete details on the use and execution of this protocol, please refer to Gusenda et al.¹

Graphical abstract

Highlights

• •Recombinant production of ACPs and purification by affinity chromatography/precipitation
• •Acylation of apo-ACP with various substrates
• •Purification of acyl-ACP from holo-ACP by PEGylation and SEC
• •Analysis by urea PAGE, HPLC, and NMR spectroscopy

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Acyl carrier proteins (ACPs) are key domains in fatty acid and polyketide synthases (PKSs), shuttling intermediates to catalytic partners. Here, we present a protocol for purifying and analyzing ACPs from fatty acid synthases (FASs) and PKSs, using four model ACPs from Mus musculus, Saccharopolyspora erythraea, Streptomyces venezuelae, and Streptomyces hygroscopicus. We describe steps for recombinant ACP production in E. coli; purification via chromatography or precipitation; ACP modification (holo/acyl forms); and analysis using urea PAGE, high-performance liquid chromatography (HPLC), and NMR spectroscopy.

Before you begin

To work with the ACPs from mFAS or PKSs, a plasmid encoding the target ACP is required. The following protocols were tested and optimized with the ACP from murine FAS and three ACPs from PKS systems: DEBS 3 ACP₅, PikAIII ACP₅ and RapC ACP₁₄. The pairwise sequence identity between these PKS ACPs and the mFAS ACP lies in the range of 25%–31%, while they share 46%–55% sequence identity with each other. We note that to facilitate expression in E. coli, the genes encoding the PKS ACPs which all derive from Actinomycete species, were codon optimized using the IDT (https://eu.idtdna.com) codon optimization tool. In this context, it should be noted that expression levels in E. coli and protein solubility may strongly depend on the ACP donor organism. For functionalizing/acylating the apo-ACPs, we utilized the broad specificity phosphopantetheinyl transferase Sfp, which can be produced following the protocols available.^2,3,4

General preparations

Timing: variable

• 1.Prepare the buffers and stock solutions, as described in materials and equipment. Prepare all solutions and media using Milli-Q water or equivalent to the required total volumes.

Note: Certain stock solutions can be prepared in advance in water to make buffer preparation easier, including: 1 M KH₂PO₄, 1 M K₂HPO₄, 1 M CaCl₂, 1 M MgSO₄, 0.5 M EDTA, 1 M NEM, 1 M NAM and 10% w/v APS. All aqueous stock solutions with the exception of maleimide can be stored at room temperature (∼22°C) for as long as one year. NEM and NAM solution should be prepared fresh before usage.

Note: Read the safety data sheets as provided by the suppliers.

Use personal protective equipment (safety glasses, gloves, lab coat, etc.) and best lab practice when working with acids and bases (e.g., HCl, NaOH, acetic acid, etc.), as they can cause damage to eyes and skin. A fume hood must be employed when working with concentrated HCl. Also use personal protective equipment (safety glasses, gloves, lab coat, etc.) when working with methanol, as it is toxic and can be absorbed through skin (H225, H301, H302, H305, H311, H331, H370) and N-ethylmaleimide (NEM), as it is toxic in contact with skin and if inhaled, as well as an irritant (H300, H301, H311, H314, H317). Finally, use personal protective equipment (safety glasses, gloves, lab coat, etc.) as well as a fume hood when working with acrylamide solution, as it is toxic and cancerogenic (H301, H312, H340, H350, H361fd, H372).

• 2.Prepare and sterilize all media and equipment for the work with bacteria cultures, as indicated in materials and equipment.

Note: The protocol steps that describe handling of bacteria must be carried out in a GMO (genetically modified organism) suitable laboratory. Equipment and material that has been in contact with GMOs must be sterilized with suitable disinfectant or by autoclaving prior to disposal. According to site-specific safety regulations, it may be necessary to wear gloves when working with DNA and hazardous chemicals.

Construct generation

Timing: 3 days

• 3.Insert the gene encoding the acyl carrier protein into a pET22b(+) vector downstream the T7 promotor using In-Fusion Cloning, following the manufacturer’s guidelines.

CRITICAL: If you wish to purify the target ACPs via Ni-NTA affinity chromatography, a C-terminal His₈-tag should be included. If you wish to purify acyl-ACP after functionalization with Sfp as described in the protocol below, an N-terminal Strep-tag should be included.

• 4.Transform Stellar chemically competent E. coli cells with the In-Fusion Cloning product, following the manufacturer’s guidelines.
• 5.Grow on LB agar plates (supplemented with 100 μg/mL ampicillin and 1% glucose) at 37°C overnight.
• 6.Transfer one colony to 20 mL LB medium (supplemented with 100 μg/mL ampicillin and 1% glucose) and incubate at 37°C overnight.
• 7.Purify the plasmid with the geneJET Plasmid-Miniprep-Kit, following the manufacturer’s guidelines.
• 8.Check the purified plasmid by sequencing and store at −18°C.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
Stellar chemically competent cells	Takara Bio	Cat#636763
BL21-Gold(DE3) competent cells	Agilent	Cat#230132
Chemicals, peptides, and recombinant proteins
Acetic acid	Merck	Cat#1.00063.1011
Acetone	VWR	Cat#20066.330
Acrylamide/bisacrylamide	Sigma-Aldrich	Cat#A3574
Ammonium chloride (15N-NH4Cl)	Cambridge Isotope Laboratories	Cat#NLM-467-10
Ammonium persulfate (APS)	AppliChem	Cat#A1032,1000
Ampicillin sodium salt	Carl Roth	Cat#K029.2
D-biotin	IBA	Cat#2-1016-005
Boric acid (H3BO3)	Carl Roth	Cat#6943.1
Bromophenol blue	Merck	Cat#1.08122.0005
Calcium chloride (CaCl2)	Carl Roth	Cat#A119.1
Cobalamin	Carl Roth	Cat# T915.2
Cobalt chloride (CoCl2)	Carl Roth	Cat#7095.1
Coomassie blue R-250	AppliChem	Cat#A1092,0100
Copper chloride (CuCl2)	Carl Roth	Cat#1L60.1
Deuterium oxide (D2O)	Deutero	Cat#00506
Dipotassium hydrogen phosphate (K2HPO4 · 3 H2O)	Thermo Scientific	Cat#205930025
Disodium hydrogen phosphate dihydrate (Na2HPO4 · 2 H2O)	Carl Roth	Cat#4984.1
DNase I	AppliChem	Cat#A3778,0500
Ethanol	VWR	Cat#20823.327
Ethylenediaminetetraacetic acid (EDTA)	Merck	Cat#1.08418.1000
Glycerol	Carl Roth	Cat#7533.4
Glycine	Merck	Cat#1.04201.1000
His60 Ni Superflow resin	Takara	Cat#635662
2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid (HEPES)	Sigma-Aldrich	Cat#H4034
Imidazole	Carl Roth	Cat#3899.3
Iron (III) chloride (FeCl3)	Carl Roth	Cat# 5192.1
Isopropanol	Sigma-Aldrich	Cat#33539
Isopropyl-β-D-thiogalactopyranoside (IPTG)	Carl Roth	Cat#2316.5
LB medium	Carl Roth	Cat#X964.2
Magnesium chloride hexahydrate	Carl Roth	Cat#2189.1
Magnesium sulfate (MgSO4)	Carl Roth	Cat#P027.1
Maleimide-PEG5K-OH (MPEG)	Sigma-Aldrich	Cat#63187
Manganese chloride (MnCl2)	Carl Roth	Cat#4320.1
Methanol	VWR	Cat#20864.320
N-(2-aminoethyl) maleimide (NAM)	Sigma-Aldrich	Cat#56951
N-ethylmaleimide (NEM)	Sigma-Aldrich	Cat#E3876
Niacin	Carl Roth	Cat#3815.1
Nickel sulfate (NiSO4)	Carl Roth	Cat#T111.2
Potassium chloride	Merck	Cat#1.04936.1000
Potassium dihydrogen phosphate (KH2PO4)	Carl Roth	Cat#P018.2
Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS)	Sigma-Aldrich	Cat#613150
Sodium chloride	VWR	Cat#27810.295
Sodium dihydrogen phosphate	Carl Roth	Cat#4984.1
Sodium molybdate (Na2MoO4)	Carl Roth	Cat#0274.3
Sodium selenite (NaSeO3)	Carl Roth	Cat#1E0Y.1
Strep-TactinXT 4Flow	IBA Lifesciences	Cat#2-5030-025
Tetramethyl (TEMEDA)	VWR	Cat#443083G
Thiamine	Carl Roth	Cat#T911.1
Trifluoro acetic acid	VWR	Cat#153112E
Tris(hydroxymethyl)aminomethane (Tris)	Carl Roth	Cat#0188.4
Urea	AppliChem	Cat#A2514,5000
Zinc sulfate (ZnSO4)	Carl Roth	Cat#2293.3
Critical commercial assays
GeneJET miniprep kit	Thermo Fisher Scientific	Cat#K0502
In-Fusion snap assembly master mix	Takara Bio	Cat#638945
Recombinant DNA
pET22b(+) vector	Merck Millipore	Cat#69744
Software and Algorithms
ImageJ	Schneider et al.5	https://imagej.net/software/imagej/
TopSpin 4.0.9	Bruker BioSpin
NMRFAM-sparky	Lee et al.6	http://pine.nmrfam.wisc.edu/download_packages.html
Other
UltiMate 3000 HPLC system	Thermo Fisher Scientific	https://www.thermofisher.com/de/de/home/industrial/chromatography/liquid-chromatography-lc/hplc-uhplc-systems/ultimate-3000-hplc-uhplc-systems.html
Mini-PROTEAN gel system or similar	Bio-Rad	Cat#1658000
NanoDrop 2000c spectrometer or similar	Thermo Fisher Scientific	Cat#ND-2000C
Cytiva S200 10/300 size exclusion column	Cytiva	Cat# 28990944
Centrifugal filters Amicon 10K 0.5 mL	Merck	Cat#UFC501096
Centrifugal filters Amicon 10K 4 mL	Merck	Cat#UFC801096
Centrifugal filters Amicon 10K 15 mL	Merck	Cat#UFC901096
Centrifugal filters Ultrafree 0.22 μm	Merck	Cat#UFC30GVNB
Standard 3 mm NMR tube “Economy”	Hilgenberg	Cat#2001749
Syringe filters ReliaPrep 0.45 μm	Ahlstrom-Munksjö	Cat#760517

Materials and equipment

ACP buffer

Reagent	Final concentration	Amount
Aqueous KH2PO4 (1 M)	20 mM	20 mL
Aqueous K2HPO4 (1 M)	30 mM	30 mL
KCl	200 mM	14.9 g
Glycerol	10% v/v	100 mL
EDTA (0.5 M)	1 mM	2 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.0 with HCl and/or KOH.

Store at room temperature (∼22°C) up to 2 months.

Acylation buffer, 10× stock

Reagent	Final concentration	Amount
HEPES	50 mM	120 g
NaCl	200 mM	116 g
MgCl2· 6 H2O	10 mM	20 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.0 with HCl and/or NaOH.

Store at 4°C up to one year.

Ampicillin solution, 1000× stock

Reagent	Final concentration	Amount
Ampicillin	100 mg/mL	10 g
ddH2O	N/A	Up to 100 mL
Total	N/A	100 mL

Sterilize using a syringe filter.

Store at −20°C for up to one month.

IPTG solution

Reagent	Final concentration	Amount
Isopropyl-β-D-thiogalactopyranoside (IPTG)	1 M	23.8 g
ddH2O	N/A	Up to 100 mL
Total	N/A	100 mL

Sterilize using a syringe filter.

Store at −20°C for up to one month.

M9 medium

Reagent	Final concentration	Amount
Aqueous CaCl2 (1 M)	0.1 mM	0.1 mL
Aqueous MgSO4 (1 M)	2 mM	2 mL
M9 salts (10×)∗	N/A	100 mL
Glucose (20% w/v)	1% w/v	50 mL
15N-NH4Cl	18 mM	1 g
Trace elements (5000×)∗	N/A	0.2 mL
Vitamins (2000×)∗	N/A	0.5 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Sterilize by autoclaving.

Store at room temperature (∼22°C) for up to 3 days.

∗See recipe below.

M9 salts, 10× stock

Reagent	Final concentration	Amount
Na2HPO4 · 2 H2O	530 mM	94.5 g
KH2PO4	220 mM	30 g
NaCl	86 mM	5 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Sterilize by autoclaving.

Store at room temperature (∼22°C) for up to 2 months.

Trace elements solution, 5000×

Reagent	Final concentration	Amount
FeCl3	50 mM	81 mg
MnCl2	10 mM	14 mg
CoCl2	2 mM	48 mg
Na2MoO4	2 mM	48 mg
H2BO3	2 mM	12 mg
NiSO4	2 mM	53 mg
ZnSO4	10 mM	179 mg
CuCl2	2 mM	34 mg
NaSeO3	2 mM	35 mg
ddH2O	N/A	Up to 100 mL
Total	N/A	100 mL

Vitamin solution, 2000 x

Reagent	Final concentration	Amount
Thiamine	100 mM	506 mg
Biotin	100 mM	367 mg
Nicotinic acid	100 mM	185 mg
Cobalamin	10 mM	203 mg
Total	N/A	15 mL

PEGylation buffer

Reagent	Final concentration	Amount
Tris	50 mM	6.1 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.0 with HCl and/or NaOH.

Store at room temperature (∼22°C) for up to 2 months.

Ni wash buffer

Reagent	Final concentration	Amount
Aqueous KH2PO4 (1 M)	20 mM	20 mL
Aqueous K2HPO4 (1 M)	30 mM	30 mL
KCl	200 mM	14.9 g
Glycerol	10% v/v	100 mL
Imidazole	30 mM	1.8 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.0 with HCl and/or KOH.

Store at room temperature (∼22°C) for up to 2 months.

Ni elution buffer

Reagent	Final concentration	Amount
Aqueous KH2PO4 (1 M)	20 mM	20 mL
Aqueous K2HPO4 (1 M)	30 mM	30 mL
KCl	200 mM	14.9 g
Glycerol	10% v/v	100 mL
Imidazole	300 mM	18 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.0 with HCl and/or KOH.

Store at room temperature (∼22°C) for up to 2 months.

Potassium phosphate solution, 10× (for TB medium)

Reagent	Final concentration	Amount
KH2PO4	170 mM	23.14 g
K2HPO4 · 3 H2O	720 mM	164.32 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.5 with HCl and/or KOH and sterilize by autoclaving.

Store at room temperature (∼22°C) for up to 2 months.

Strep wash buffer

Reagent	Final concentration	Amount
Aqueous KH2PO4 (1 M)	20 mM	20 mL
Aqueous K2HPO4 (1 M)	30 mM	30 mL
Glycerol	10% v/v	100 mL
Aqueous EDTA (0.5 M)	1 mM	2 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Adjust to pH 7.0 with HCl and/or KOH.

Store at room temperature (∼22°C) for up to 2 months.

Strep elution buffer

Reagent	Final concentration	Amount
Aqueous KH2PO4 (1 M)	20 mM	20 mL
Aqueous K2HPO4 (1 M)	30 mM	30 mL
Glycerol	10% v/v	100 mL
Aqueous EDTA (0.5 M)	1 mM	2 mL
Biotin	50 mM	1.22 g
ddH2O	N/A	Up to 100 mL
Total	N/A	100 mL

Adjust to pH 7.0 with HCl and/or KOH.

Store at room temperature (∼22°C) for up to 2 weeks.

TB medium

Reagent	Final concentration	Amount
Tryptone	N/A	12 g
Yeast extract	N/A	24 g
Glycerol	5% v/v	5 mL
Potassium phosphate (10x)	∼90 mM	100 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Prepare the TB medium base with all components except the potassium phosphate in 900 mL water, and sterilize by autoclaving.

Prepare the potassium phosphate (10×) separately and add just prior to inoculation.

Store at room temperature for up to 3 days.

Urea PAGE destaining solution

Reagent	Final concentration	Amount
Ethanol	10% v/v	100 mL
Acetic acid	10% v/v	100 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Store at room temperature (∼22°C) for up to one year.

Urea PAGE gel buffer, separating gel, 3×

Reagent	Final concentration	Amount
Tris	1.13 M	1.5 g
Urea	7.5 M	4.5 g
ddH2O	N/A	Up to 10 mL
Total	N/A	10 mL

Adjust to pH 9.0 with HCl and/or KOH.

Store at −20°C for up to 6 months. Redissolve the urea well by shaking/vortexing during thawing.

Urea PAGE gel buffer, stacking gel, 3×

Reagent	Final concentration	Amount
Tris	0.38 M	0.5 g
Urea	7.5 M	4.5 g
ddH2O	N/A	Up to 10 mL
Total	N/A	10 mL

Adjust to pH 6.8 with HCl and/or KOH.

Store at −20°C for up to 6 months. Redissolve the urea well by shaking/ vortexing during thawing.

Urea PAGE loading dye, 2×

Reagent	Final concentration	Amount
Bromophenol blue	1% w/v	100 mg
Tris	62.5 mM	76 mg
Urea	2 M	1.2
Glycerol	30% v/v	3 mL
Aqueous maleimide (NEM/NAM) (1 M)	10 mM	100 μL
ddH2O	N/A	Up to 10 mL
Total	N/A	10 mL

Adjust to pH 6.8 with HCl and/or KOH.

Store in 1 mL aliquots at −20°C for up to 6 months.

Urea PAGE running buffer

Reagent	Final concentration	Amount
Tris	25 mM	3.1 g
Glycine	192 mM	14.4 g
ddH2O	N/A	Up to 2 L
Total	N/A	2 L

Adjustment of pH not needed.

Store at −4°C for up to 6 months.

Urea PAGE separating gel

Reagent	Final concentration	Amount
Acrylamide/Bis-acrylamide (30%)	∼15%	5 mL
Gel buffer	N/A	3.5 mL
ddH2O	N/A	2 mL
TEMEDA	N/A	10.5 μL
aqueous APS (10% w/v)	N/A	32 μL
Total	N/A	10.5 mL

Prepare the mixture and mix well. Immediately after addition of TEMEDA and APS, pour 4.65 mL into the Mini-PROTEAN gel system (Bio-Rad, cat. no. 1658000). Leave it to solidify at room temperature (∼22°C) for approximately 30 min.

Read Safety Note.

Urea PAGE stacking gel

Reagent	Final concentration	Amount
Acrylamide/Bis-acrylamide (30%)	∼5%	500 μL
Gel buffer	N/A	1 mL
ddH2O	N/A	1.5 mL
TEMEDA	N/A	3 μL
aqueous APS (10% w/v)	N/A	9 μL
Total	N/A	3 mL

Prepare the mixture and mix well. Pour the solution onto the separating gel. Leave the gel to solidify at room temperature (∼22°C) for approximately 30 min.

Read Safety Note.

Urea PAGE staining solution

Reagent	Final concentration	Amount
Coomassie Blue R-250	0.1% w/v	1 g
Methanol	50% v/v	500 mL
Acetic acid	10% v/v	100 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Store at room temperature (∼22°C) for up to 6 months.

Read Safety Note.

Step-by-step method details

Recombinant expression of ACP in E. coli and purification of non-functionalized apo-ACP

Timing: 3 days

The following protocol describes the production of non-functionalized apo-ACP by recombinant expression of ACP encoding sequences from plasmids in E. coli with subsequent lysis of cells (troubleshooting 1−3).

• 1.Express the target genes in the chosen strain of E. coli, and harvest the crude extract after cell disruption and centrifugation, according to standard procedures.
  Example protocol.

  • a.
    Prepare a preculture of BL21 Gold (DE3) cells containing the target ACP gene on a pET22b vector in 20 mL LB medium (supplemented with 100 μg/mL ampicillin and 1% glucose).
  • b.
    Incubate overnight (∼17 h) at 37°C.
  • c.
    Transfer the total of 20 mL preculture to 1 L TB medium (supplemented with 100 μg/mL ampicillin).

    Note: To express ¹⁵N labeled protein for NMR studies, use for example M9 medium instead of TB. Use glycerol-free buffers for the purification steps.

  • d.
    Grow the culture at 37°C and 140 rpm to an optical density at 600 nm (OD₆₀₀) of 0.6–0.8.
  • e.
    Cool the culture below 20°C and induce expression by addition of 250 μL sterile 1 M IPTG solution (final [IPTG] = 0.25 mM).
  • f.
    Incubate 16 h at 20°C and 140 rpm.
  • g.
    Centrifuge the cultures at 45,000g for 20 min.
  • h.
    Discard the supernatant and resuspend the cells in 20 mL Ni wash buffer.
  • i.
    Add a small amount (∼0.5 mg) of DNase I and 60 μL of 0.5 mM EDTA to the suspension.
  • j.
    Disrupt the cells using a French press.
  • k.
    Centrifuge the disrupted cell mixture at 40,000g for 1 h, and collect the supernatant as a crude extract.
• 2.
  Purify the crude extract by Ni-NTA affinity chromatography or alternatively isopropanol precipitation (see Step 3). The purification via affinity chromatography might produce higher yields (see expected outcomes).

  • a.
    Transfer the crude extract to 5 mL His60 Ni Superflow Resin. Collect the flowthrough for analysis via SDS-PAGE.
  • b.
    Wash the resin with 15 mL Ni wash buffer. Collect the wash fraction for analysis by SDS-PAGE (Figures 1 and S1).
  • c.
    Elute the ACP by applying 7.5 mL Ni elution buffer to the resin. Collect the fraction, and prepare a sample for analysis by SDS-PAGE (Figure 1).
  • d.
    Repeat the elution step once (if the two elution fractions both contain pure protein, they can be combined).

Note: Coomassie Blue staining of SDS-PAGE gels can disproportionately underestimate the proportion of ACP, as smaller proteins often stain less intensely than their higher-molecular-weight counterparts.

• 3.
  Alternatively purify the crude extract by precipitation in isopropanol and acetone

  • a.
    Precipitate non-ACP proteins by adding an equivalent volume of cold isopropanol (−20°C) to the crude extract.
  • b.
    Centrifuge the mixture for 30 min at 15,000g, 4°C. Collect the supernatant. Prepare a sample for analysis with SDS-PAGE (Figures 2 and S2).

    Note: Centrifuge tubes vary in their resistance towards isopropanol. Therefore, it is prudent to employ single-use tubes.

  • c.
    To rebuffer the sample, precipitate the ACP from the 50% isopropanol solution using 4 volume equivalents of cold acetone (−20°C).
  • d.
    Centrifuge for 10 min at 15,000g, 4°C. Collect the precipitate.
  • e.
    Resolubilize the precipitate in ACP buffer and remove insoluble particles by centrifugation (10 min, 15,000g, 4°C).

    Note: It is possible to further purify the construct using Strep-Tactin resin (if a Strep-tag present) or size exclusion chromatography (SEC), as described in the sections “acylation of apo-ACP” and “size exclusion of PEGylated holo-ACP”, respectively. For comprehensive structure verification after precipitation and solubilization see step 13–14 under “Analysis of functionalized acyl-ACP with Urea PAGE, HPLC and NMR spectroscopy”.

Figure 1.

Ni-affinity chromatography

SDS-PAGE analysis, showing the steps of Ni-affinity chromatography purification. Crude extract refers to the lysate after breaking the cells with a French Press and centrifugation (step 1k), flow-through, wash and elution refer to the fractions of Ni-NTA purification (steps 2a−d). The gel shows the results for mFAS ACP (12.1 kDa) and DEBS 3 ACP₅ (15.2 kDa). Further examples can be found in the supplementary document (Figure S1).

Figure 2.

Precipitation in organic solvents

SDS-PAGE analysis, showing the steps of purification by precipitation in isopropanol and acetone. Crude extract refers to the lysate after breaking the cells with French Press and centrifugation (step 1k), isopropanol supernatant refers to the supernatant after isopropanol precipitation and centrifugation (step 3b), acetone supernatant and precipitate refer to the acetone precipitation from isopropanol solution (step 3d, e). The gel shows the results for mFAS ACP (12.1 kDa) and DEBS 3 ACP₅ (15.2 kDa). Further examples can be found in the supplementary document (Figure S2).

Acylation of apo-ACP to generate crypto-ACP from respective CoA esters

Timing: 5 h

This protocol describes the acylation of apo-ACP and the subsequent purification of acyl-ACP to yield highly pure acyl-ACP (based on strep-tagged ACP) (troubleshooting 4−7).

• 4.
  Perform the acylation reaction.

  • a.
    Prepare 1 mL solution of 30 μM Sfp, 3 mM acyl-CoA, and 600 μM apo-ACP in acylation buffer in a 1.5 mL microcentrifuge tube on ice.
  • b.
    Incubate for 20 min at 37°C and 400 rpm in a thermoshaker.

Note: Absorption at 260 nm can be measured at NanoDrop to determine concentrations of acyl-CoA solutions in the μM range (ε₂₆₀ = 16.4 mM⁻¹cm⁻¹). To measure concentration of solutions in mM range, 1:1000 dilutions should be prepared and measured, since devices like NanoDrop work best in this range. The concentration of the undiluted solution can be derived from the concentration of the diluted sample.

Make sure to provide for sufficient amounts of Mg²⁺ in the reaction mix (close to 1 mM). When protein- and acyl-CoA solutions are present in buffers without Mg²⁺, the reaction mix can be filled up e.g., with 2x acylation buffer, to reach sufficient final concentration of buffer components.

CRITICAL: Use highly pure acyl-CoA samples (i.e. free from coenzyme A contaminant). Phosphopantetheine groups from any free coenzyme A will be preferably transferred to the apo-ACP by Sfp, and will thus yield holo-ACP instead of the desired acyl-ACP. In this case, the protocol “Purification of acyl-ACP from holo-ACP impurities” should be followed.

• 5.
  Purify the acyl-ACP from the reaction mixture using Strep-tag based affinity chromatography.

  • a.
    Transfer the reaction mixture directly to a column with 5 mL Strep-TactinXT 4Flow resin. Collect the flow-through, and analyze a sample by SDS-PAGE.
  • b.
    Wash the resin with 15 mL of Strep wash buffer. Collect the wash fraction, and analyze a sample by SDS-PAGE (Figures 3 and S3).
  • c.
    Elute the acyl-ACP with 12.5 mL of Strep elution buffer. Collect the elution fraction, and analyze a sample by SDS-PAGE.
• 6.
  Re-buffer the elution fraction using a 3 kDa or 10 kDa cutoff Amicon filter (alternatively: use the precipitation protocol with acetone, as described in Strep 3c−e).

  • a.
    Concentrate the elution fractions to 1.5 mL, add 3 mL ACP buffer and centrifuge for about 10 min at 5,000g (until 1.5 mL).
  • b.
    Repeat this step at least 4 times.

Note: The rebuffering of ACP reduces the biotin concentration considerably, which facilitates subsequent concentration measurements using the NanoDrop. Aim for a concentration of approximately 15 mg/mL.

• 7.Snap-freeze the samples in liquid nitrogen and store at −80°C.

Figure 3.

Strep-tag-based affinity chromatography

SDS-PAGE analysis, showing the steps of Strep-tag-based affinity chromatography purification of the acylation reaction mixture. Reaction mix refers to the solution of apo-ACP, Sfp and acyl-CoA (step 4), while flow-through, wash and elution refer to the fractions of the affinity chromatography (step 5a−c). The gel shows the results for DEBS 3 ACP₅ (15.2 kDa). Additional examples (e.g., HPLC-traces) can be found in the supplementary document and previous work. (Figures S8 and S9 of the referenced work).¹

Purification of acyl-ACP from holo-ACP impurities

Timing: 5 h

The acylation of apo-ACP can sometimes result in holo-ACP impurities (due e.g., to the presence of CoA in the commercial acyl-CoA, or to hydrolysis of the acyl group from acyl-ACP). This protocol describes the purification of acyl-ACP by PEGylation of holo-ACP, followed by purification by SEC. (troubleshooting 8)

• 8.
  Perform the PEGylation reaction (Figures 4 and S4).

  • a.
    Adjust the concentration of the ACP solution to 500 μM by diluting it in ACP buffer.
  • b.
    Add Maleimide-PEG5K-OH (MPEG) equivalent to a 2-molar excess (e.g., 2.5 mg in 500 μL) directly into the ACP solution at room temperature (∼22°C), mix by pipetting up and down or inverting the tube.
  • c.
    Incubate reaction mixture in a thermoshaker at 37°C for 1 h, 400 rpm.
• 9.
  Separate the acyl-ACP and PEGylated holo-ACP by SEC (Figure 4).

  • a.
    Prepare 500 mL of ACP buffer for SEC usage by filtration with 0.2 μm bottle-top filters and degassing via 20 min ultrasonication under vacuum.
  • b.
    Prepare the sample for SEC by filtration using 0.2 μm centrifugal filters.
  • c.
    Run the chromatography at 4°C according to the size exclusion manual. Procedure for Cytiva S200 10/300 column:

    • i.
      Equilibrate the column with 2 CV of ACP buffer (50 mL) at a flow rate of 0.4 mL/min at 4°C.
    • ii.
      Equilibrate a 2 mL sample loop with 10 mL of ACP buffer. Load the loop with your sample and inject the sample onto the column with 4 mL of ACP buffer.
    • iii.
      Run the chromatography at 0.4 mL/min for 1.2 CV (30 mL).

      Note: Two major peaks are expected. The peak of lower retention time and thus greater size or hydrodynamic radius is expected to be the PEGylated holo-ACP, whereas the peak of higher retention time is expected to be unmodified acyl-ACP. Collect and pool the fractions corresponding to either of these peaks.

  • d.
    Concentrate the pure acyl-ACP to an appropriate concentration for downstream application (e.g., 30 mg/mL), using 3 kDa or 10 kDa cut-off centrifugal filters.

    Note: The buffer and sample preparation can be adjusted to the specific size exclusion system that is used.

Figure 4.

PEGylation of holo-ACP

(A) The size exclusion chromatogram of a holo-ACP/ mal-ACP (mFAS) mixture, which was supplemented with MPEG (step 9c iii).

(B) Urea-PAGE analysis (steps 10, 11) of the PEGylation of holo-ACP. Fractions F1 and F2 of the SEC of mFAS ACP from Figure 4A are shown. The reaction mixture of holo-ACP, mal-ACP and the mixture of holo- and mal-ACP is shown for DEBS 3 ACP₅. Further examples can be found in the supplementary document (Figure S4).

Analysis of functionalized acyl-ACP with urea PAGE, HPLC, and NMR spectroscopy

Timing: 4–5 h/method

The following protocol shows how to identify the functional state of ACPs (apo, holo, crypto) using Urea PAGE and HPLC and the comprehensive structural analysis by NMR is described. Depending on the substrate bound to ACP, loading dye supplemented with NEM or NAM, respectively, enables separation during Urea PAGE. Examples can be found in Figure 4, the supplementary document and in previous work.¹

Note: It is recommended to use Urea PAGE with NEM for apo-, malonyl- and succinyl-ACP; Urea PAGE with NAM for saturated fatty acid-, β-hydroxy butyryl- and β-oxo butyryl-ACP; and HPLC for small to medium chain fatty acid- and crotonyl-ACP; NMR structure analysis can be employed regardless of the cargo. If available, mass spectrometry can be employed to identify the functional state of ACPs. Destaining of Urea PAGE gels for more than one night can cause the intensity of the protein bands to weaken. The use of a molecular weight marker is not necessary or useful, because the electrophoretic mobility does not correlate with molecular weight − analyzing the relative mobility is sufficiently informative.

• 10.
  Prepare and run Urea PAGE.

  • a.
    Prepare an ACP sample solution at 25 μM in 10 μL (dilute your stock with water if necessary). Include apo and holo reference samples, if available.
  • b.
    Add 10 μL loading dye (containing either NEM or NAM) and mix well.
  • c.
    Incubate the mixture for 10 min at room temperature (∼22°C).
  • d.
    Load 10 μL sample onto the gel.
  • e.
    Run the electrophoresis at 70 V for 15 min and subsequently at 200 V for 60−90 min (until the dye exits the gel).
• 11.
  Stain the resulting gel.

  • a.
    Place the gel in a plastic box. Soak the gel in fresh Urea PAGE staining solution for 1 h–16 h.
  • b.
    To destain the background, soak the gel in Urea PAGE destaining solution. Include a folded paper towel in the box for higher destaining efficiency.
  • c.
    Scan the gel image with a gel imaging device or a common document scanner.

Note: The appropriate analysis method can vary depending on the cargo that is loaded onto the ACP.

• 12.
  Alternatively: Analyze your sample with HPLC.

  • a.
    Prepare the buffers by diluting 0.1% m/v trifluoroacetic acid (TFA) in water and acetonitrile (ACN), respectively. Filter the solutions using Durapore filters.
  • b.
    Prepare a 50 μM ACP sample and dilute 25 μL of sample with 25 μL aqueous 0.1% TFA solution.
  • c.
    Centrifuge the sample at 20,000g for 10 min to separate dust and other particles.
  • d.
    Transfer 25 μL of the solution to an HPLC vial and place it in the HPLC sampler. Inject 10−15 μL sample during the analysis.
  • e.
    Run the analysis as following:

Solvent A = H₂O, 0.1% TFA.

Solvent B = ACN, 0.1% TFA.

Flow rate 0.5 mL/min.

0−3 min, ramp up to 39% B;

3−15 min, ramp up to 42% B; 15−16 min, ramp up to 60% B;

16−18 min, 60% B;

18−19 min, ramp down to 20% B;

19–21 min, 20% B.

• 13.
  To verify the structural integrity/fold of ACPs, the domains can be analyzed by NMR. For this, record ¹H 1D NMR spectrum of uniformly ¹⁵N-labeled ACP that was expressed in M9 medium (optional).

  • a.
    Prepare 200 μL of NMR sample containing 150 μM ACP (concentration can be lowered to 50–100 μM if protein yield is low), 1 mM sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as external reference and 5% D₂O in ACP buffer.
  • b.
    Transfer the NMR sample to a 3 mm NMR tube and record ¹H 1D NMR spectra, using a gradient-assisted excitation sculpting pulse sequence for water suppression.⁷
  • c.
    Process and analyze the data using TopSpin 4.0.9 (or another version).

Note: If the ACP is well-folded, amide proton resonances will be observed between 6 and 11 ppm (Figure 5).

Figure 5.

NMR spectra

(A) Amide proton resonance region in the ¹H-1D NMR spectrum of uniformly ¹⁵N-labeled apo-ACP (step 13).

(B) ¹H, ¹⁵N-BEST-TROSY NMR spectra (step 14) of uniformly ¹⁵N-labeled apo-ACP in supplemented ACP buffer (50 mM HEPES, 200 mM NaCl, 10 mM MgCl₂, 1 mM DSS, 5% D₂O). The spectra show the structural integrity of ACP as indicated by the identical peak pattern. Further examples, including structure verification after precipitation, can be found in the supplementary document (Figure S5).

• 14.
  Record ¹H-¹⁵N 2D BEST-TROSY spectrum and assign resonances, if available.

  • a.
    Use the same NMR sample from step 13 to record a ¹H-¹⁵N BEST-TROSY⁸ spectrum with a number of scans (ns) = 32.
  • b.
    Process and analyze the data using TopSpin 4.0.9 (or another version).
  • c.
    Export the spectrum to NMRFAM-sparky⁶ and compare it to the resonance assignment that is given in the BMRB solution NMR assignment data for the respective ACP, if available (Figure 5: Type I FAS ACP).

Note: Chemical shifts may deviate slightly due to different buffer conditions or measurement temperature compared to the BMRB data.⁹ It is also possible that individual resonances are absent from the spectrum as a consequence of protein dynamics. If the solution NMR structure of the ACP is not published yet, assignments cannot be transferred. If you find the peaks distributed across the range of 6 and 11 ppm, protein quality is satisfactory.

Expected outcomes

The expected results for apo-ACP expression and Ni-NTA affinity purification, acylation of apo-ACP, purification of acyl-ACP and HPLC analysis as well as Urea PAGE with NEM and NAM are shown in previous work.¹

The anticipated yield of apo-ACP from recombinant expression after His-purification is approximately 120 mg protein/L TB culture (Step 2) whereas the yield following isopropanol precipitation is approximately 40 mg/L (Step 3) (as determined for mFAS ACP). When cells are cultivated in M9 medium, the expected yields of mFAS and PKS ACPs are in the range of 8–25 mg/L. The purity of the isolated apo-ACP can vary, which depends on the expression quantity. However, the purity after the first purification (Step 2 or 3) improves upon secondary purification (Step 5 or 9). The conversion of apo-ACP to acyl-ACP during phosphopantetheinylation by Sfp is quantitative. The ratio of acyl- to holo-ACP depends on the initial quantity of contaminating CoA, as well as the rates of hydrolysis of the acyl-CoA and the resulting acyl-ACP. The expected yields after subsequent purification with affinity chromatography are approximately 50% (12 mg acyl-ACP per 24 mg apo-ACP). Purification of apo-ACP by isopropanol precipitation can lead to improved purity (e.g., from 14−25% initial purity to 27−99% final purity (Figure 1A)). The purification of acyl- from holo-ACP by PEGylation and subsequent SEC is expected to improve purity (e.g., from 45% initial purity to 90% final purity, as shown for malonyl-ACP (Figure 4)).

Limitations

The protocols were established for the mFAS ACP, DEBS 3 ACP₅, PikAIII ACP₅ and RapC ACP₁₄. Adaptation of the protocols to acyl carrier proteins of other systems might be necessary. Purification using isopropanol precipitation is based on the high overexpression of ACP relative to other E. coli proteins. If the expression is less successful, the purity will not be drastically improved by this protocol.

Troubleshooting

Problem 1

Step 1: A mixture of apo- and holo-ACP is generated upon recombinant expression in M9 medium.

Potential solution

Ensure the correct supplementation with trace minerals and vitamins. An unoptimized proportion can lead to production of an apo-/holo-ACP mixture.

Problem 2

Step 3: ACP supernatant becomes cloudy after isopropanol precipitation.

Potential solution

The solubility of ACP in 2-propanol is highly temperature-dependent. Warming the solution to room temperature (∼22°C) should clear the solution.

Problem 3

Step 3: Precipitation with acetone leads to phase separation.

Potential solution

Precipitation in acetone is dependent on the buffer. High salt content could lead to phase separation and incomplete precipitation. ACP buffer and acylation-buffer are suitable for acetone precipitation.

Problem 4

Step 4: Acylation efficiency is low.

Potential solution

Phosphate can cause precipitation of magnesium ions which are necessary for Sfp activity during long term storage of acylation buffer. Use non-complexing buffer agents instead of phosphate.

Problem 5

Step 4: Hydrolysis of acyl-CoAs or acyl-ACPs is high.

Potential solution

Certain additives such as the reducing agent DTT promote hydrolysis of the thioester. DTT is generally not needed. In case disulfides of holo-ACP are observed, reduce the disulfide bond with the reducing agent TCEP. Remove the TCEP thereafter via centrifugation or dialysis.

Problem 6

Step 4: Long-chain CoAs precipitate in assay solution.

Potential solution

Large concentrations of C10-CoA (and higher) might not be soluble. Nonetheless, as soon as the ACP and Sfp are present in solution, small quantities of soluble acyl-CoAs are transferred to the ACP. The resulting acyl-ACPs exhibit higher solubility than their more hydrophobic acyl-CoA counterparts. Ultimately due to the law of mass action, the acyl-CoAs will eventually solubilize.

Problem 7

Step 4: Long-chain ACPs precipitate in assay solution.

Potential solution

C10-ACP (and higher) are not soluble at high concentrations (>100 μM) and low temperature. Warming up to room temperature (∼22°C) may help.

Problem 8

Step 8: Acyl-ACP amount is dramatically reduced after PEGylation.

Potential solution

MPEG should not be used at too great an excess, as it promotes the side reaction of the maleimide warhead with amines. This spontaneous chemistry likely results in non-stochiometric modification of both holo-ACP and acyl-ACP. Additionally, a decrease in acyl-ACP yield following PEGylation and SEC purification can be due to the protein loss during injection, fractionation etc.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Prof. Martin Grininger, grininger@chemie.uni-frankfurt.de.

Technical contact

Technical questions on executing this protocol should be directed to the technical contact, Christian Gusenda, gusenda@biochem.uni-frankfurt.de.

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets/code.

Acknowledgments

We thank E. Helfrich and his lab, especially Milena Breitenbach, for assistance in using LC-MS instrumentation. Support for this work was provided by the German Chemical Industry Fund with a Kekulé Scholarship awarded to C.G., as well as the German Research Foundation (DFG grant GR3854/10-1 to M.G.). This work has further been supported by the DFG (GRK 1986 – Complex Light Control and project number 531012774) and the state of Hesse (BMRZ). Work in K.J.W.’s lab was funded by the Université de Lorraine and the CNRS.

Author contributions

C.G. and M.G. designed the experiments. C.G. performed the purification, functionalization, and analysis with PAGE and HPLC of murine ACP and analyzed the resulting data. S.B. performed the purification, functionalization, and analysis with PAGE of PKS ACPs and analyzed the resulting data. I.B. performed the NMR analysis of murine ACP and analyzed the resulting data. C.G., S.B., I.B., and M.G. wrote the protocol, and K.J.W. edited it. M.G., B.C., K.J.W., and H.S. supervised the study.

Declaration of interests

The authors declare no competing interests.