Purification and Identification of Antimicrobial Protein from the Green Alga Tetraspora Sp. CU2551

Summary

This protocol describes a workflow for investigating intact antimicrobial proteins using multiple techniques, including optimization of protein extraction buffers, algal cell disruption, protein fractionation by ion-exchange chromatography, protein separation by SDS-PAGE, and antimicrobial activity assays. The active protein was subsequently confirmed and identified using LC-MS/MALDI-TOF analysis.

Abstract

Antimicrobial resistance (AMR) is an escalating global health threat, necessitating the discovery of novel antimicrobial agents. Algae-derived proteins and peptides have gained increasing attention for their bioactive potential; however, standardized protocols for investigating antimicrobial peptides (AMPs) in green algae, particularly Tetraspora sp. CU2551, remain limited. This protocol describes a step-by-step workflow for the isolation, purification, and characterization of antimicrobial intact proteins from the green alga Tetraspora sp. CU2551. The procedure integrates optimized protein extraction, chromatographic fractionation, electrophoretic separation, antimicrobial activity screening, and protein identification. Protein extraction conditions are first optimized to reduce background inhibitory effects originating from buffer components. When the identity of the active peptide is unknown, a pull-down assay is applied to assess protein binding across different ion-exchange resins and to guide the selection of an appropriate purification matrix. DEAE-Sepharose ion-exchange chromatography is a suitable method for enriching antimicrobial protein fractions. All fractions are systematically evaluated for antimicrobial activity and analyzed by SDS-PAGE. The protein band corresponding to the highest antimicrobial activity, along with a distinct electrophoretic profile, is excised and subjected to LC-MS/MALDI-TOF analysis for protein identification. The workflow is further complemented by in silico analyses to predict antimicrobial peptide-related properties using publicly available bioinformatic tools. This protocol provides a versatile framework for antimicrobial protein discovery and can be readily adapted to other algal species and related biotechnological applications.

Introduction

Antimicrobial resistance (AMR) represents an increasing global health challenge¹, highlighting the importance of ongoing strategies to identify novel antimicrobial agents from natural sources. Although there is increasing research on the antimicrobial properties of proteins and peptides derived from algae^2,3. Most studies focus on peptide- or hydrolysate-based approaches⁴, and standardized methods to preserve and evaluate the activity of whole algal proteins remain limited⁵. This article describes a method aimed at isolating, purifying, and evaluating intact proteins from the green alga Tetraspora sp. CU2551 for antimicrobial activity.

The protocol outlines a step-by-step process encompassing the extraction of whole proteins, fractionation by ion-exchange chromatography, assessment of antibacterial properties, gel-based protein separation, protein identification by mass spectrometry, and prediction of antimicrobial peptides using in silico techniques. The effectiveness of antibacterial agents is assessed using standard agar diffusion and liquid growth inhibition tests on typical Gram-positive and Gram-negative bacterial strains^4,6. This approach enables the connection of identified antimicrobial activity to specific protein fractions or electrophoretic bands, rather than relying on digested peptide mixtures or solely on computational predictions that may fail to identify antimicrobial proteins or AMP-like sequences outside canonical database definitions^7,8.

Following this protocol enables readers to reliably obtain bioactive protein fractions containing potential antimicrobial protein sequences, suitable for future validation and functional assessment. Representative findings indicate the identification of bioactive fractions containing both known and unknown protein candidates. This protocol provides a versatile and practical framework for identifying antimicrobial substances from intact proteins obtained from algae and other biological sources.

Protocol

This study did not involve human participants, animals, or vertebrate subjects. All experimental procedures were conducted in accordance with the institutional biosafety and research ethics guidelines of the School of Science, King Mongkut's Institute of Technology Ladkrabang (KMITL), Thailand. All microbial experiments were performed under Biosafety Level 2 (BSL-2) laboratory conditions following WHO and the national biosafety standard⁹.

Wear appropriate personal protective equipment, including laboratory coats, gloves, and face masks, throughout all experimental procedures. Collect and dispose of chemical and silver-containing wastes according to institutional hazardous waste management guidelines prior to sending them to a licensed waste management company.

Prepare concentrated stock solutions of hazardous chemicals, including sodium dodecyl sulfate, β-mercaptoethanol, and formaldehyde-containing silver staining reagents, in a chemical fume hood. Conduct experiments using low working concentrations and small volumes on the laboratory bench in accordance with standard laboratory safety practices.

1. Algal culture

NOTE: Maintain aseptic conditions throughout algal cultivation.

1. Starter culture preparation
   1. Prepare sterile TAP medium (pH 7.0)¹⁰ and dispense 150 mL into sterile 500 mL Erlenmeyer flasks.
   2. Inoculate the medium with a 1% (v/v) inoculum overnight culture of Tetraspora sp. CU2551 using aseptic technique in a Class I biosafety cabinet.
   3. Incubate the cultures in a light-equipped shaking incubator at 35 °C, 120 rpm, and 45.9 µmol/m²/s for 48 h. Use the fully grown culture as a starter for scale-up culture.
2. Scale-up culture and cell harvesting
   1. Transfer 25 mL of starter culture into 275 mL of fresh sterile TAP medium. Incubate the cultures under the same conditions for 48 h.
   2. Harvest the algal cells by centrifugation at 3,800 × g for 15 min at 25 °C.
   3. Wash the cell pellets once with sterile distilled water and centrifuge again at 3,800 × g for 15 min at 25 °C.
   4. Remove the supernatant completely and store the algal cell pellets at −20 °C until further processing.

2. Lysis buffer formulation and background inhibition screening

CAUTION: Handle SDS and β-mercaptoethanol with rubber or nitrile gloves. Open the concentrated β-mercaptoethanol bottle in a chemical fume hood.

1. Lysis buffer preparation
   1. Prepare the reference lysis buffer¹¹ consisting of 10 mM Tris-HCl (pH 9.0) supplemented with 0.1 % w/v SDS and 38 mM β-mercaptoethanol.
      NOTE: Critical step: Optimize SDS and β-mercaptoethanol concentrations to avoid false-positive antimicrobial activity.
   2. Prepare full factorial sets of lysis buffer by varying sodium dodecyl sulfate (SDS) (0, 0.01, 0.05, or 0.1% w/v) and β-mercaptoethanol (0, 0.5, 1, or 2 mM), generating 16 formulations.
      NOTE: The compositions of all tested lysis buffer formulations are summarized in Table 1. Add β-mercaptoethanol immediately before use.
2. Background antimicrobial screening
   1. Use a paper punch to prepare 6 mm disks from No. 42 Whatman paper. Apply 5 µL of each lysis buffer formulation onto the paper disk and perform disk diffusion assays according to CLSI guidelines⁶. Incubate the assay plates at 37 °C for 20 h and assess inhibition zones.
      1. Test formulations against Bacillus subtilis TISTR 1248, Staphylococcus aureus TISTR 746, Escherichia coli TISTR 074, and Pseudomonas aeruginosa TISTR 2370.
   2. Select the formulation showing no inhibitory activity for subsequent protein extraction.

3. Protein extraction

NOTE: Maintain samples at low temperature during sonication and centrifugation.

1. Thaw frozen algal pellets (from step 1.2.5) at 37 °C and refreeze at −20 °C. Repeat freeze-thaw cycles (-20 °C / 37 °C) 3x.
2. Add optimal lysis buffer (10 mM Tris-HCl, pH 9.0, 0.01% SDS, 2 mM β-mercaptoethanol) to a final volume of 5 mL and keep samples on ice throughout downstream processing. Disrupt the cells by probe sonication (25 kHz, 500 W, 40 % amplitude, 1 s on/off, 5 min).
3. Add Protease Inhibitor Cocktail according to the manufacturer's instructions and mix gently.
4. Centrifuge lysates at 3,800 × g for 15 min at 4 °C and collect the supernatant as crude protein extract.
5. Clarify the extracts by centrifugation at 10,000 × g for 10 min at 4 °C. Collect and store the supernatants at 4 °C.

4. Antimicrobial activity evaluation

1. Disk diffusion assays
   1. Culture pathogenic bacteria (Bacillus subtilis TISTR 1248, Staphylococcus aureus TISTR 746, Escherichia coli TISTR 074, and Pseudomonas aeruginosa TISTR 2370) on nutrient agar at 37 °C for 16-18 h.
   2. Using a sterile aluminum loop, scrape the colonies from agar surfaces and resuspend in 0.9% (w/v) NaCl. Adjust bacterial suspensions to 0.5 McFarland standard and swab evenly onto Mueller-Hinton agar plates.
   3. Place sterile paper disks onto inoculated agar surfaces and apply 5 µL of test samples. Incubate the plates at 37 °C for 16-20 h and measure inhibition zones using a Vernier caliper.
      NOTE: Maintain equal spacing among all disks.
2. MIC and MBC determination
   1. Prepare bacterial suspensions at 2 × 10⁶ CFU/mL in 4x Mueller-Hinton broth.
   2. Pipette 50 µL of the bacterial suspensions and 100 µL of the serially diluted protein samples into 96-well plates and adjust all wells to a final volume of 200 µL using 10 mM Tris-HCl buffer (pH 7.5).
   3. Use 5 µL of streptomycin (50 µg) as a positive control. Use 10 mM Tris-HCl (pH 7.5) and 0.9 % (w/v) NaCl as negative controls.
   4. Incubate plates at 37 °C for 20 h and determine MIC as the lowest concentration without visible bacterial growth.
   5. Plate aliquots from clear wells onto Mueller-Hinton agar to determine MBC.
3. Bacterial growth curve determination
   1. Prepare bacterial suspensions at 1 × 10⁶ CFU/mL in 2x Mueller-Hinton broth.
   2. Incubate 100 µL of bacterial culture with 100 µL of protein samples in a 96-well plate at 37 °C for 20 h.
   3. Use 5 µL of streptomycin (50 µg) as a positive control. Use sterile distilled water, 10 mM Tris-HCl (pH 7.5), and 500 mM NaCl in 10 mM Tris-HCl (pH 7.5) as controls.
   4. Measure OD₆₀₀ every 30 min using a microplate reader with orbital intermittent shaking (2 min before measurement).
   5. Calculate growth curves and specific growth rates.
      NOTE: High protein concentrations may interfere with optical density measurements.

5. Pull-down assay and protein purification

1. Pull-down assay
   1. Equilibrate DEAE-Sepharose resin with 10 mM Tris-HCl buffer (pH 8.0) or CM-Sepharose resin with 10 mM Phosphate buffer (pH 7.0).
   2. Mix 100 µL of equilibrated resin with 500 µL of crude protein extract, then add 100 µL of the corresponding equilibration buffer (10 mM Tris-HCl, pH 8.0 for DEAE; 10 mM phosphate buffer, pH 7.0 for CM). Incubate with gentle inversion (10 inversions).
   3. Centrifuge briefly, collect the supernatant, and evaluate antimicrobial activity by disk diffusion assay.
   4. Select the working resin (DEAE-Sepharose). For pH optimization, equilibrate resin with 10 mM Tris-HCl at pH 7.0, 7.5, 8.0, 8.5, or 9.0 and follow steps 5.1.1-5.1.3.
   5. Select the pH condition showing antimicrobial activity for ion-exchange chromatography.
2. Ion-exchange chromatography-based protein purification
   1. Pack DEAE-Sepharose resin into a glass chromatography column (16 mL bed volume) and equilibrate with 10 mM Tris-HCl buffer (pH 7.5) at 25 °C for 10 column volumes.
   2. Load 15 mL of crude protein extract (section 3.6) at a flow rate of 8 mL/min.
   3. Wash the column with equilibration buffer until absorbance at 280 nm falls below 0.05.
   4. Elute bound proteins using a linear 0-500 mM NaCl gradient in 10 mM Tris-HCl (pH 7.5) over 500 mL at 8 mL/min.
   5. Collect 5 mL fractions, monitor absorbance at 280 and 220 nm, and keep fractions on ice. Store fractions at 4 °C until antimicrobial screening and protein analysis.

6. SDS-PAGE and silver staining

NOTE: Prepare silver staining solutions in a chemical fume hood and wear appropriate personal protective equipment. Prepare all solutions freshly.

1. Separate protein fractions using 12% SDS-PAGE¹² and visualize bands by silver staining¹³.
2. Excise target protein bands and submit samples for MALDI-TOF MS analysis.
3. Identify proteins by database searching against NCBInr.

7. Antimicrobial peptide prediction

1. Retrieve candidate protein sequences from NCBInr analysis.
2. Evaluate antimicrobial potential using publicly available AMP prediction tools (e.g., DBAASP, APD3, AMPScanner, CAMPR4) and databases.
3. Extract prediction outputs, including antimicrobial probability scores, predicted classification, and physicochemical properties (net charge, hydrophobic residue content, GRAVY index [ExPASy ProtParam], and amphipathicity).
4. Integrate computational results across platforms to prioritize candidate antimicrobial proteins for downstream validation.
   NOTE: In silico predictions support candidate selection and do not constitute definitive classification of antimicrobial peptides.

8. Statistical testing

1. Perform all experiments in biological triplicate. Analyze data using one-way ANOVA followed by Duncan's multiple range test or appropriate post hoc analyses at a 95% confidence level.

Results

The developed intact-protein-based workflow, integrating optimized protein extraction, ion-exchange fractionation, antimicrobial screening, and protein identification, successfully enabled the detection of bioactive protein fractions from Tetraspora sp. CU2551. This protocol is designed to minimize background artifacts while preserving native protein integrity, allowing antimicrobial activity to be directly associated with defined chromatographic fractions and electrophoretic bands.

O...

Discussion

To obtain reliable antimicrobial readouts using this protocol, several critical steps must be carefully controlled, particularly during protein extraction and early bioactivity screening. Optimizing the lysis buffer is essential, as background inhibition from buffer components is a frequent source of false-positive results in antimicrobial protein screening. The antimicrobial activity observed with the original buffer formulation¹¹ highlights this issue, given that SDS can disrupt bacterial membra...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acrylamide/Bis-acrylamide Solution	Bio-Rad Laboratories	1610156	For SDS-PAGE gel preparation Category: Chemical
Alcohol Lamp / Bunsen Burner	Various	—	For aseptic technique Category: Instrument
AMPScanner	George Mason University (Veltri et al.)	—	Deep learning-based AMP prediction tool Category: Software / Database
Antimicrobial Peptide Prediction Tools (DBAASP, APD3, AMPscanner, CAMPR4)	Various academic sources	—	In silico AMP analysis Category: Software / Database
APD3	Wang Lab, University of Nebraska	—	Antimicrobial Peptide Database Category: Software / Database
Autoclave	Tomy	—	Sterilization of media and materials Category: Instrument
Biosafety Cabinet Class I/II	Various	—	Aseptic handling Category: Instrument
BioTool 2.0	Bruker Daltonics	—	Tool for visualizing MS spectra and peptides Category: Software
Bradford Reagent	Merck	B6916	Protein quantification Category: Chemical
CAMPR4	Indian Institute of Technology Bombay	—	Collection of antimicrobial peptides Category: Software / Database
Centrifuge, Eppendorf 5415R Refrigerated Microcentrifuge	Eppendorf	—	Microcentrifugation Category: Instrument
Centrifuge, Thermo Scientific Megafuge 8R	Thermo Fisher Scientific	—	Large-volume centrifugation Category: Instrument
Chromatography Column (Glass)	Various	—	Ion-exchange chromatography Category: Glassware
CM-Sepharose Resin	Merck	CEF100	Cation-exchange resin Category: Chemical (Resin)
Cotton Swab	Various	—	Bacterial inoculation Category: Plasticware
Cuvette, Quartz	Hellma Analytics	—	UV spectrophotometry Category: Glassware
DBAASP	Institute of Bioorganic Chemistry (Russia)	—	Database of antimicrobial activity and peptide structures Category: Software / Database
DEAE-Sepharose Resin	Merck	DEFF100	Anion-exchange resin Category: Chemical (Resin)
Erlenmeyer Flask, 100 mL	Various	—	Algal cultivation Category: Glassware
Erlenmeyer Flask, 500 mL	Various	—	Algal cultivation Category: Glassware
Escherichia coli TISTR 074	TISTR, Thailand	—	Test microorganism Category: Biological material
ExPASy ProtParam	Swiss Institute of Bioinformatics	—	Online tool for computing physicochemical properties of proteins Category: Software / Database
Formaldehyde	Merck	1.04002	Silver staining Category: Chemical
Freezer (-20 °C)	Various	—	Sample storage Category: Instrument
Gel Electrophoresis System (Mini-PROTEAN Tetra)	Bio-Rad Laboratories	—	SDS-PAGE Category: Instrument
Glass Test Tube (16 × 150 mm)	Pyrex / Duran	—	Sample handling Category: Glassware
Gloves, Nitrile	Various	—	Personal protection Category: Plasticware
Halt Protease Inhibitor Cocktail (100x)	Thermo Fisher Scientific	87786	Protease inhibition Category: Chemical
Incubator	Various	—	Bacterial culture Category: Instrument
Incubator Shaker	Various	—	Algal culture Category: Instrument
Inoculating loop	Any laboratory supplier	—	Microbial transfer Category: Consumable
KCl	Merck	P5405	Buffer preparation Category: Chemical
MALDI-TOF Mass Spectrometer (Reflex IV)	Bruker Daltonics	—	Protein identification Category: Instrument
MASCOT	Matrix Science	—	Search engine for protein identification via MS data Category: Software / Database
MASCOT 2.2	Matrix Science	—	Protein identification Category: Software
Measuring Cylinder	Pyrex	—	Volume measurement Category: Glassware
Microcentrifuge Tube (1.5 mL)	-	—	Sample processing Category: Plasticware
Microcentrifuge Tube (2.0 mL)	-	—	Sample processing Category: Plasticware
Micropipette (100–1000 µL)	-	—	Liquid handling Category: Instrument
Micropipette (1–10 µL)	-	—	Liquid handling Category: Instrument
Micropipette (1–5 µL)	-	—	Liquid handling Category: Instrument
Micropipette (1–5 mL)	-	—	Liquid handling Category: Instrument
Micropipette (20–200 µL)	-	—	Liquid handling Category: Instrument
Microplate Reader	BioTek	—	OD600 measurement Category: Instrument
Mueller–Hinton Agar/Broth	Various	—	Antimicrobial assays Category: Medium
Nanodrop / Biodrop Spectrophotometer	Various	—	Protein measurement Category: Instrument
NCBInr database	National Center for Biotechnology Information	—	Non-redundant protein database Category: Software / Database
Nutrient Agar	-	—	Bacterial culture Category: Medium
Paper Disk (6 mm)	Whatman No.42	—	Disk diffusion assay Category: Plasticware
Petri Dish	Various	—	Microbial culture Category: Plasticware
pH Meter	Mettler Toledo	—	pH adjustment Category: Instrument
Phosphate Buffer (10 mM, pH 7.0)	-	—	Chromatography buffer Category: Buffer
Probe Sonicator (VCX500)	Sonics & Materials Inc.	—	Cell disruption 500 W, 20 kHz, with probe Category: Instrument
Refrigerator (4 °C)	Various	—	Sample storage Category: Instrument
Silver Nitrate	Sisco Research Laboratories	94118	Silver staining Category: Chemical
Sodium Chloride (NaCl)	Merck	S9888	Elution buffer Category: Chemical
Sodium Dodecyl Sulfate (SDS)	Bio-Rad Laboratories	11667289001	Lysis and PAGE Category: Chemical
Spectrophotometer	Shimadzu	—	Absorbance measurement Category: Instrument
Spin-down Centrifuge	Eppendorf	—	Quick centrifugation Category: Instrument
Staphylococcus aureus TISTR 746	TISTR, Thailand	—	Test microorganism Category: Biological material
Streptomycin	Merck	S9137-25G	Positive control Category: Chemical
Tetraspora sp. CU2551	KMITL Culture Collection	—	Algal strain Category: Biological material
Tris Acetate Phosphate (TAP) Medium	-	—	Algal culture Category: Medium
Tris-HCl	Merck	—	Buffer preparation Category: Buffer
Vernier Caliper	Various	—	Zone measurement Category: Instrument
Water Bath	Various	—	Temperature control Category: Instrument
XMASS	Bruker Daltonics	—	MALDI-TOF mass spectrometry software Category: Software
α-Cyano-4-hydroxycinnamic acid (CHCA)	Bruker Daltonics	—	MALDI matrix Category: Chemical