Protein Purification Steps Research Articles

Recombinant strains expressing oxalate decarboxylase efficiently and environmental characteristics of their life cycle assessment

Oxalate decarboxylase has garnered significant attention due to its wide-ranging applications, particularly as an effective demulsifier for oil pollution remediation. However, the accumulation of oxalate decarboxylase by strain is insufficient, and the acquisition of necessary nutrients and energy for biosynthesis also poses challenges to the environment. Therefore, it is crucial to assess the strain's capacity and environmental burden in order to guide future developments and ensure sustainable scale-up. To enhance the protein yield of oxalate decarboxylase from Bacillus mojavensis XH1 (Bacm OxdC), a specialized biodemulsifier, our study investigated the properties of Bacm OxdC and improved its expression efficacy in Escherichia coli from an environmentally friendly perspective. By employing a promoter tandem strategy and optimizing the 5′ untranslated region, recombinant strains C3 and C5 were constructed, resulting in 1.22-fold and 6.90-fold increases in Bacm OxdC expression levels compared to the initial strain C1. The bioprocess of strain C5 achieved a life cycle assessment (LCA)-based environmental impact value of 322.75, representing the lowest environmental impact observed thus far. Improvement strategies included reducing protein purification steps and electricity consumption, which can further mitigate negative environmental impacts and carbon emissions. This study offers a technical strategy for large-scale production of oxalate decarboxylase while attaining green and low-carbon objectives for biological demulsifiers.

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein.

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes.

Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (GstCeuE and PthCeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with CjCeuE, the analogous protein of Campylobacter jejuni. Upon immobilization, the GstCeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, while retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles, a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilized protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilized ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilization of redox-reversible ArMs facilitates their "catch-and-release" assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.

Mammalian Antigen Display for Pandemic Countermeasures.

Pandemic countermeasures require the rapid design of antigens for vaccines, profiling patient antibody responses, assessing antigen structure-function landscapes, and the surveillance of emerging viral lineages. Cell surface display of a viral antigen or its subdomains can facilitate these goals by coupling the phenotypes of protein variants to their DNA sequence. Screening surface-displayed proteins via flow cytometry also eliminates time-consuming protein purification steps. Prior approaches have primarily relied on yeast as a display chassis. However, yeast often cannot express large viral glycoproteins, requiring their truncation into subdomains. Here, we describe a method to design and express antigens on the surface of mammalian HEK293T cells. We discuss three use cases, including screening of stabilizing mutations, deep mutational scanning, and epitope mapping. The mammalian antigen display platform described herein will accelerate ongoing and future pandemic countermeasures.

Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads.

Nanobodies as imaging agents and drug conjugates have shown great potential for cancer diagnostics and therapeutics. However, site-specific modification of a nanobody with microbial transglutaminase (mTGase) encounters problems in protein separation and purification. Here, we describe a facile yet reliable strategy of immobilizing mTGase onto magnetic beads for site-specific nanobody modification. The mTGase immobilized on magnetic beads (MB-mTGase) exhibits catalytic activity nearly equivalent to that of the free mTGase, with good reusability and universality. Magnetic separation simplifies the protein purification step and reduces the loss of nanobody bioconjugates more effectively than size exclusion chromatography. Using MB-mTGase, we demonstrate site-specific conjugation of nanobodies with fluorescent dyes and polyethylene glycol molecules, enabling targeted immunofluorescence imaging and improved circulation dynamics and tumor accumulation in vivo. The combined advantages of MB-mTGase method, including high conjugation efficiency, quick purification, less protein loss, and recycling use, are promising for site-specific nanobody functionalization and biomedical applications.

Extended Biocatalytic Halogenation Cascades Involving a Single-Polypeptide Regeneration System for Diffusible FADH2.

Flavin-dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C-H positions because they are characterised by high regioselectivity, while requiring only FADH2 , halide salts, and O2 . Their use in combined crosslinked enzyme aggregates (combiCLEAs) together with an NADH-dependent flavin reductase and an NADH-regeneration system for the preparative halogenation of tryptophan and indole derivatives has been previously described. However, multiple cultivations and protein purification steps are necessary for their production. We present a bifunctional regeneration enzyme for two-step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to co-expression with various flavin-dependent Trp-halogenases and enables carrier-free immobilisation as combiCLEAs from a single cultivation for protein production and the preparative synthesis of halotryptophan. The scalability of this system was demonstrated by fed-batch fermentation in bench-top bioreactors on a 2.5 L scale. Furthermore, the inclusion of a 6-halotryptophan-specific dioxygenase into the co-expression strain further converts the halogenation product to the kynurenine derivative. This reaction cascade enables the one-pot synthesis of l-4-Cl-kynurenine and its brominated analogue on a preparative scale.

A simple preparation of prestained molecular markers for electrophoresis using inexpensive and readily available proteins

Protein electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) is one of the most commonly performed procedures in biochemical laboratories. It requires the use of molecular weight (MW) markers as an internal technical control and to determine the migration rate of a particular protein. In this work, we describe a simple method for preparing “homemade” prestained protein markers using readily available cow's milk and chicken egg white proteins without the need of any major protein purification step, and produce prestained MW markers ranging from 19 to 98 kDa.

Evaluation of the Synergistic Effect of LL-37 and Oncorhyncin II Recombinant Proteins on Staphylococcus aureus Under In Vitro Conditions

Background: The treatment of Staphylococcus aureus infections has become a public health crisis due to the extensive development of antimicrobial resistance. Antimicrobial peptides (AMPs) have been introduced as promising naturally-derived antimicrobial alternatives to antibiotics. LL-37 and oncorhyncin II are 2 AMPs with notable proven antibacterial effects. Objectives: This study aimed to produce recombinant LL-37 and oncorhyncin II and investigate their synergistic effects on S. aureus (ATCC25923). Methods: The synthetic genes of LL-37 and oncorhyncin II were individually ligated into the pET32a expression vector. Transformed pET32a was introduced into Escherichia coli BL21 as an expression host. The protein expression and purification steps were optimized, and the biological effectiveness of the peptides was evaluated by assessing the minimum inhibitory concentration (MIC), time-kill, and growth kinetic tests against S. aureus. Results: The MIC assay confirmed the effective antibacterial performances of LL-37 and oncorhyncin II against S. aureus at 30.6 and 47.93 µg/mL, respectively. The peptides’ synergistic activity was validated by the checkerboard method. A combination of LL-37 and oncorhyncin II at 2 × MIC showed a sharp decline of the viable cells with over 3-time reductions in log 10 colony-forming units (CFU)/mL within the first 5 hours. The growth kinetic results confirmed the high effectiveness of the peptides’ combination in eliminating the bacterial inoculum turbidity by 50% reduction during the first hour of exposure. Conclusions: The produced recombinant LL-37 and oncorhyncin II showed effective antimicrobial function against S. aureus. The synergistic performance of the peptides was repeatedly confirmed through checkerboard, time-kill, and growth kinetic assays.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase (LHyal) is a hyperactive hyaluronic acid (HA) hydrolase that belongs to the hyaluronoglucuronidase family. Traditionally, LHyal is extracted from the heads of leeches, but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides. However, at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps. Moreover, the enzyme is deactivated and removed after single use. To solve this problem, we developed a recyclable LHyal biocatalyst using a yeast surface display (YSD) system. After screening and characterization, we found that the cell wall protein Sed1p displayed stronger anchoring to the P. pastoris cell wall than other cell wall proteins. By optimizing the type and length of the linkers between LHyal and Sed1p, we increased the activity of enzymes displayed on the P. pastoris cell wall by 50.34% in flask cultures. LHyal-(GGGS)6-Sed1p activity further increased to 3.58 × 105 U mL−1 in fed-batch cultivation in a 5 L bioreactor. Enzymatic property analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme. Moreover, displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times. YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification process for HA oligosaccharides. Thus, this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Protein Extraction and Purification by Differential Solubilization.

The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this process. Selectively altering the solubility of a protein is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of a polypeptide. Precipitation of proteins, released from cells upon lysis, is often used to concentrate a protein of interest before further purification steps (e.g., ion exchange chromatography, size exclusion chromatographyetc).Recombinant proteins may be expressed in host cells as insolubleinclusion bodies due to various influences during overexpression. Such inclusion bodies can often be solubilized to be reconstituted as functional, correctly folded proteins.In this chapter, we examine strategies for extraction/precipitation/solubilization of proteins for protein purification. We also present bioinformatic tools to aid in understanding a protein's propensity to aggregate/solubilize that will be a useful starting point for the development of protein extraction, precipitation, and selective re-solubilization procedures.

A simple and high -performance immobilization technique of membrane protein from crude cell lysate sample for a membrane-based immunoassay application

ABSTRACT Membrane proteins are difficult to be extracted and to be coated on the substrate of the immunoassay reaction chamber because of their hydrophobicity. Traditional method to prepare membrane protein sample requires many steps of protein extraction and purification that may lead to protein structure deformation and protein dysfunction. This work proposes a simple technique to prepare and immobilize the membrane protein suspended in an unprocessed crude cell lysate sample. Membrane fractions in crude cell lysate were incorporated with the large unilamellar vesicle (LUV) that was mainly composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) before coating in the polystyrene plate by passive adsorption technique. Immunofluorescence staining and the Enzyme-Linked Immunosorbent Assay (ELISA) examination of a strictly conformation-dependent integral membrane protein, Myelin Oligodendrocyte Glycoprotein (MOG), demonstrate that LUV incorporated cell lysate sample obviously promotes MOG protein immobilization in the microplate well. With LUV incorporation, the dose–response curve of the MOG transfected cell lysate coating plate can be 2–9 times differentiated from that of the untransfected cell lysate coating plate. The LUV incorporated MOG transfected cell lysate can be efficiently coated in the microplate without carbonate/bicarbonate coating buffer assistance.

A living material platform for the biomineralization of biosilica.

Nature has a vast array of biomineralization mechanisms. The commonly shared mechanism by many living organisms to form hardened tissues is the nucleation of mineral structures via proteins. Living materials, thanks to synthetic biology, are providing many opportunities to program cells for many functionalities. Here we have demonstrated a living material system for biosilicification. Silaffins are utilized to synthesize silicified cell walls by one of the most abundant organism groups called diatoms. The R5 peptide motif of the silaffins is known for its ability to precipitate silica in ambient conditions. Therefore, various studies have been conducted to implement the silicification activity of R5 in different application areas, such as regenerative medicine and tissue engineering. However, laborious protein purification steps are required prior to silica nanoparticle production in recombinant approaches. In this study, we aimed to engineer an alternative bacterial platform to achieve silicification using released and bacteria-intact forms of R5-attached fluorescent proteins (FP). Hence, we displayed R5-FP hybrids on the cell surface of E. coli via antigen 43 (Ag43) autotransporter system and managed to demonstrate heat-controllable release from the surface. We also showed that the bacteria cells displaying R5-FP can be used in silicification reactions. Lastly, considering the stimulating effect of silica on osteogenic differentiation, we treated human dental pulp stem cells (hDPSCs) with the silica aggregates formed via R5-FP hybrids. Earlier calcium crystal deposition around the hDPSCs was observed. We envision that our platform can serve as a faster and more economical alternative for biosilicification applications, including endodontics.

PRODUCTION OF ANTI-LACTOFERRIN ANTIBODIES AND THEIR APPLICATION IN ANALYSIS OF THE TEAR FLUID IN HEALTH AND CORNEAL INJURIES

Lactoferrin is a ubiquitous and multifunctional protein, which has antimicrobial and immunomodulatory activities. Lactoferrin plays an important role in the maintenance of ocular health. The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury. Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P<0.05. Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons (P<0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation. Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way.

Structural Determinants of Blue to Red Fluorescent Protein Conversion

The color, brightness, and stability of a fluorescent protein are dictated by its amino acid sequence. Attempts to alter the physical properties of fluorescent proteins have focused on random amino acid changes directed at positions known to be a part of or near the fluorophore in the protein. In this study TagBFP and TagRFP, which differ by 13 amino acid mutations, are compared to understand which amino acids are critical for the fluorophore's color. Several site-directed mutants were created, and the in vivo fluorescence of the 17 different mutant proteins were evaluated. The fluorescence of the proteins was measured in vivo, skipping the time-consuming step of protein purification since the fluorescence was not affected by purification. Nearly all mutants exhibit blue fluorescence. The color conversion from blue to red required the mutation of a full set of six amino acids that contact the fluorophore.

Development and application of metal chelate-epoxy bifunctional loofah sponge for the purification and immobilization of recombinant trehalose synthase

The preparation and application of metal chelate-epoxy bifunctional loofah sponge for the selective adsorption and covalent immobilization of a His-tagged trehalose synthase were reported. The adsorption of the His-tagged trehalose synthase on the metal chelate loofah sponge was found to follow the Langmuir isotherm model with Co2+ ion exhibiting an adsorption capacity of 1.183 mg/g and a superior selectivity over the other metal ions tested. Bifunctional loofah sponge, designated as BiFLS57, with an adsorption capacity of 0.639 mg/g and an [epoxy groups]/[metal chelate groups] ratio of 0.75 was used for enzyme immobilization. The ideal incubation conditions for the selective adsorption and covalent immobilization of the His-tagged trehalose synthase were 1.0 h at 4 °C and 16 h at 30 °C, respectively. The immobilized enzyme thus prepared exhibited an enzyme activity of 3.99 U/g with a 93% immobilization yield and an 84% global activity yield. The immobilized enzyme prepared with the metal chelate-epoxy bifunctional loofah sponge BiFLS57 exhibited superior thermostability and operational stability in a repeated-batch process, retaining 96% of its initial activity after 20 cycles. The employment of the metal chelate-epoxy bifunctional loofah sponge can significant facilitate enzyme immobilization processes by consolidating protein purification and enzyme immobilization steps.

Semi-stable Production of Bovine IL-4 and GM-CSF in The Mammalian Episomal Expression System.

IntroductionGranulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) are cytokines widely used in ex vivo monocyte differentiation experiments, vaccine formulations and disease treatment. The aim of this study was to produce recombinant bovine GM-CSF and IL-4 in an episomal expression system that conserves the postransductional modification of the native proteins and to use the products to differentiate bovine monocytes into dendritic cells.Material and MethodsThe recombinant proteins rGM-CSF and rIL-4 were expressed in PEAKrapid CRL-2828 human kidney cells, ATCC CRL-2828. The functional activity of the recombinant cytokines was monitored by registering morphological changes in bovine monocytes and assessing the expression of CD14 upon incubation with them.ResultsBoth recombinant proteins were detected in the cell culture supernatant of transfected cells. Culture supernatants of transfected cells induced in bovine monocytes morphological changes that resemble macrophages or dendritic cells. In addition, bovine cells treated with rGM-CSF and rIL-4 showed reduced expression of the macrophage surface marker CD14 compared with untreated cells. This effect indicates the expected differentiation. The expression of the cytokines was stable after many successive cell passages and a freeze/thaw cycle.ConclusionsThe semi-stable mammalian episomal expression system used in this study allowed us to easily produce functional bovine rGM-CSF and rIL-4 without the need for protein purification steps.

Teaching the Principles of DNA cloning, Recombinant Protein Expression, and Purification using a Purple Chromoprotein

The Freshman Research Initiative (FRI) program at the University of Texas at Austin offers first-year students access to cutting edge research in science. The Biobricks for Molecular Machines FRI stream focuses on training first-year undergraduates (with little to no lab experience) in basic molecular and biochemical skills in their first 9-weeks in the stream. We use the purple chromogenic protein (Meffcp_GBR22), derived from Montipora efflorescens, to allow visual confirmation of various molecular cloning and recombinant protein expression and purification steps, which highly enhances the learning experience of undergraduates. By conducting this lab curriculum in a research-based setting, students learn to troubleshoot and examine sources of error by experiencing the repetitive nature of research where results are not always as expected and each lab module is linked the next. In addition to hands-on experiences, students also learn the important skills of analyzing and presenting their experimental results in bi-weekly laboratory reports. The robust laboratory modules described here have been tested and tried for the past decade and can be easily modified to incorporate any gene of interest. Further students apply the lab techniques learned on research projects that are in collaboration with other faculty labs at the university. With the use of common molecular and biochemical equipment found in basic teaching wet labs, this curriculum can be easily adapted to any university/college or advanced high school biotechnology courses.

A set of simple methods for detection and extraction of laminarinase

A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

Preliminary Study of Buffer Ratio in Protein Extraction from Placental Cotyledons of Kedah-Kelantan Cattle

Protein extraction is a preliminary step of protein purification which mainly focus on maximization of total protein yield. The heterogeneous properties cause diversification of protein; therefore, there is no absolute protocol in protein extraction. The ratio of buffer gives different protein concentrations in different types of mammalian tissues, and this condition leads to the study of optimization of buffer ratio to obtain a better total protein yield. The objectives of this study were to compare the total protein yield based on three different ratios of buffer used. The phosphate buffer saline (PBS), radioimmunoprecipitation assay (RIPA) buffer, and RIPA buffer with the addition of protease inhibitor (Pi) were used with the ratios of 1:1, 1:3, and 1:5. Fetal cotyledons removed from the placenta have undergone mechanical disruption, incubation, sonication, and centrifugation. The supernatant was retained and quantified with Bradford assay to determine the total protein yield based on the standard curve of bovine serum albumin (BSA). There was a statistically significant different between buffer ratio (p<0.5) in RIPA and RIPA with addition of protease inhibitor buffers. RIPA buffer with the ration of 1:1 gave the best total protein yield (194.880±15.089 mg/g). As a conclusion, there was a significant interaction between buffer types and have greatly enhanced the total protein yield obtained from placental cotyledons of Kedah-Kelantan cattle.

Adsorption of Phenolic Compounds from an Aqueous By-product of Sunflower Protein Extraction/Purification by Macroporous Resins

In this study, adsorption of phenolic compounds from an aqueous by-product of sunflower protein isolate production was investigated. Phenolic compounds in this by-product (ultrafiltration permeate of protein purification step) were almost exclusively CGA (mainly 5-CQA isomer). Five different macroporous resins including XAD4, XAD7, XAD16, XAD1180, and HP20 were screened for CGA capture. XAD16 had the best massic adsorption capacities (15.32 ± 0.04 mg/g), while XAD7 had a better surface adsorption capacity (0.027 ± 0.00146 mg/m²). CGA adsorption on both resins followed the pseudo-second-order kinetic model with a similar intra diffusional pattern. Adsorption isotherms of the two resins better fitted the Langmuir model with Qmax for XAD7 and XAD16 of 0.054 and 0.040 (mg/m²), respectively. The adsorption process of phenolic compounds revealed to be exothermic, physical adsorption, and spontaneous. Better adsorption results were observed at 25°C. Maximal CGA desorption ratio was observed from 70% (v/v) ethanol. The high values were reached with both the resins (88.09 ± 0.13 and 86.16 ± 0.32% for XAD7 and XAD16, respectively). The CGA purity in the desorption phase was surprisingly high (77.56 ± 0.99% and 74.59 ± 0.12% for XAD7 and XAD16, respectively).