Auxin Extraction and Purification Based on Recombinant Aux/IAA Proteins
© The Author(s). 2017
Received: 24 June 2016
Accepted: 12 December 2016
Published: 13 January 2017
Indole-3-acetic acid (IAA) extraction and purification are of great importance in auxin research, which is a hot topic in the plant growth and development field. Solid-phase extraction (SPE) is frequently used for IAA extraction and purification. However, no IAA-specific SPE columns are commercially available at the moment. Therefore, the development of IAA-specific recognition materials and IAA extraction and purification methods will help researchers meet the need for more precise analytical methods for research on phytohormones.
Since the AUXIN RESISTANT/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) proteins show higher specific binding capability with auxin, recombinant IAA1, IAA7 and IAA28 proteins were used as sorbents to develop an IAA extraction and purification method. A GST tag was used to solidify the recombinant protein in a column. Aux/IAA proteins solidified in a column have successfully trapped trace IAA in aqueous solutions. The IAA7 protein showed higher IAA binding capability than the other proteins tested. In addition, expression of the IAA7 protein in Drosophila Schneider 2 (S2) cells produced better levels of binding than IAA7 expressed in E. coli.
This work validated the potential of Aux/IAA proteins to extract and purify IAA from crude plant extracts once we refined the techniques for these processes.
KeywordsIAA Extraction and Purification Binding Capability Aux/IAA SPE
As one of the most important categories of phytohormones, auxin contributes to virtually all aspects of plant growth and development [1, 2]. Thus, phytohormone quantification is very important. Currently, IAA is extracted from plant tissues and then highly purified using standardized protocols [3, 4]. Exposure to light, heat and oxygen can cause the degradation of IAA during sample preparation and purification, as IAA is not very stable in an aqueous environment. Thus, an IAA tracer labelled with radioactivity is used to estimate the amount of recovery and correct for any loss of IAA . In the classical method, IAA extraction from plant tissues involves liquid nitrogen freezing/lyophilization, grinding/homogenization, treatment with organic solvents and the removal of solid impurities. IAA is a weak polar molecule. Thus, organic solvents with polarity close to IAA are the most effective for IAA extraction from plant tissues, as they result in greater efficiency and higher levels of extraction and recovery. However, a few recent studies used distilled water to extract IAA from plant samples . Alternate types of extraction methods have been reported in previous studies that use various organic solvents, such as ethanol, methanol, acetonitrile, chloroform, acetone, propanol, ethyl acetate and acetic acid [7–12]. Most recently, pre-cooled 80% methanol has become a universal extraction solvent for IAA and several other phytohormones because of its high efficiency during extraction and enhanced levels of recovery [6, 13].
Purification after extraction is critical because the complex metabolites in crude plant extracts can influence the accuracy of the analysis of IAA. To overcome the interference from other compounds in the extracts, a number of methods based on different principles were applied to the extraction and purification of IAA from plant tissue. Liquid–liquid extraction (LLE) and liquid–liquid microextraction (LLME), hollow fibre-based liquid–liquid–liquid microextraction (HF–LLLME) and dispersive liquid–liquid microextraction (DLLME) have been used previously for IAA purification [14–20]. Solid-phase extraction (SPE) and antibody-based immune methods have recently become widely used to extract and purify IAA [6, 21–26]. Optimized solid-phase microextraction (SPME) and double-layered SPE (DL/SPE) resulted in a higher level of recovery and better ability to remove pigments [27, 28]. SPE and SPME cartridges filled with matrix compound (sorbent) are used to extract and purify the target molecules from mixtures in the solution, as the sorbent can selectively bind certain molecule(s) based on an array of mechanisms, including adsorption, hydrogen bonding, polar and nonpolar interactions, cation/anion exchange and size exclusion . Since modern SPE/SPME techniques are usually applied online coupled with HPLC, the current emphasis involves choosing various sorbents for trapping analytes [30, 31]. Many commercial SPE/SPME columns based on different sorbents have been widely used for high-throughput phytohormone extraction and purification from crude plant extracts [22, 23, 26]. However, commercial SPE columns for multiple phytohormones, such as Sep-Pak C18, Oasis HLB, Oasis MCX and Oasis MAX, are not designed specifically for phytohormones such as IAA. Thus, they are not highly selective. Recently, molecularly imprinted polymers (MIPs) and polymer monolith microextraction (PMME) were used to improve the specific recognition capability for phytohormones [32–34]. MIPs have been widely used to detect the presence of compounds in the environment because they offer advantages such as easy, cheap and rapid preparation along with high thermal and chemical stability [35–37]. Immunoaffinity SPE sorbents, also known as immunosorbents, are based upon molecular recognition using antibodies that offer higher affinity and selectivity for the target molecule (antigen). They are suitable for extraction and purification of a single analyte from complex biological and environmental aqueous samples. Moreover, immunoaffinity chromatography (IAC) and immunoaffinity gels (IAG) have been used to purify ABA and cytokinins [38–42].
Advances in phytohormonal research require greater efficiency and increased sensitivity for the analysis of phytohormones. This should lead to advances in phytohormone extraction and purification that occur more quickly in a less complicated manner. The techniques for sample preparation are complicated, and most methods are not specially designed for IAA. In the auxin signalling pathway, auxin action is based on binding to the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) nuclear receptors. Auxin stabilizes the co-receptor complex composed of the TIR1/AFB and AUXIN RESISTANT/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) proteins that trigger the proteasome-dependent degradation of the Aux/IAA transcriptional inhibitor to release the Auxin Response Factor (ARF) factor that induces the auxin-mediated transcriptional reprogramming [43, 44]. Interestingly, TIR1, Aux/IAA proteins and co-receptors (complex of TIR1/AFB and Aux/IAA) show high affinity towards auxin both in vivo and in vitro, as this hormone acts as the molecular glue that complexes the TIR1/AFB and Aux/IAA proteins . Their auxin-specific binding characteristics can be utilized to improve the procedures for auxin extraction and purification. Therefore, we expressed AtIAA1, AtIAA7 and AtIAA28 in Escherichia coli and Drosophila Schneider 2 (S2) cells and developed a method for IAA extraction and purification using the recombinant proteins as the recognition molecules.
Chemicals and Reagents
Buffers used in the study
140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4
High pH buffer
0.1 M Tris-HCl and 0.5 M NaCl
Low pH buffer
0.1 M sodium acetate, 0.5 M NaCl
50 mM Tris-HCl and 10 mM reduced glutathione
Aux/IAAs Clone and Recombinant Vector Construction
Primer lists in vector constructions
Culture Conditions and Recombinant Protein Expression in E. Coli
Expression vectors were transformed into three E. coli strains, including BL21, Tuner and Rosetta, by electroporation. Transformants were grown at 37 °C overnight in LB medium until the OD600 reached 0.6. The cultures were diluted in fresh LB medium using a ratio of 1:50 and then grown in liquid culture at 37 °C on a shaker until the cells reached an OD600 of 0.6. Finally, the bacteria underwent another 5 h of shaking in culture to induce expression of the recombinant protein at 25 °C after adding IPTG to a concentration of 0.4 mM. The cells were collected by centrifugation and stored at −80 °C.
Culture Conditions and Recombinant Protein Expression in S2
Drosophila Schneider 2 (S2) derived from a primary culture of late stage Drosophila melanogaster embryos was purchased from Thermo Fisher Scientific Inc. (Catalogue no. R690-07). To increase the cell yield, the S2 cells were grown in Schneider’s Drosophila medium (Catalogue no. 11720-034) at 28 °C without CO2 in suspension with spinners and shake flasks according to current protocols. Split cells at a 1:2 to 1:5 ratio were diluted into new culture every 3 to 4 days when the cells reached a density of 2 to 4 × 106 cells/mL. This procedure maintained the S2 cells. The insect expression vector was stably cotransfected into S2 with pCoHygro using the calcium phosphate transfection method, and the transfectants were selected by 300 μg/mL hygromycin-B. Cell death was verified by Trypan blue staining. A 19:1 (w/w) ratio of expression vector to selection vector was used in co-transfection. Target recombinant protein was secreted into the medium and then collected by centrifugation for further purification.
Recombinant Protein Isolation and Purification
Bacterial cells were collected and resuspended in PBS (pH 7.4). Cells were disrupted by ultrasonification. Supernatant was collected for target protein purification after centrifugation at 20,000 g for 10 min at 4 °C. For insect expression, the medium was collected from 100-mL cultures by centrifugation, since the recombinant protein was secreted into the medium. Supernatant was concentrated to less than 5 mL by centrifugation at 7500 g for 10 min in Amicon Ultra-4 Centrifugal Filter Devices with a 50 kDa nominal molecular weight limit (NMWL) from Millipore at 4 °C. Condensed culture medium was diluted 5 times in PBS to prepare it for protein purification. Protein solution was added to a column pre-filled with GST Sefirose™ resin at a speed of 1 mL/min. The purified target proteins remained trapped in the column after it was washed with 5 resin volumes of PBS.
IAA Quantification by LC-MS/MS
Solution containing IAA was pumped into the column, and the IAA was trapped by the recombinant Aux/IAA proteins. Unbound molecules were washed off with 3 resin volumes of PBS. The outlet solution was collected after elution and freeze dried. The IAA sample was injected into LC-MS/MS (Shimadzu 8030 Plus) using a reverse C18 column to perform IAA determination after re-dissolving the compound in acetonitrile. Condition setting and programming for IAA detection were performed as described by Ma et al. .
Expression of Aux/IAA Proteins in E. Coli
Expression of IAA7 Protein in S2 Cells
IAA Extraction and Purification
In this study, we designed an IAA extraction and purification strategy based on the use of recombinant Aux/IAA proteins. First, we pre-filled a cartridge with GST resin to selectively bind the GST-Aux/IAA fusion proteins. This gravity column permitted isolation and purification of the target recombinant protein from the cell supernatant, as GST resin showed ideal binding activity to bioactive GST fusion proteins. Next, the aqueous sample was passed through the gravity column, and the IAA was trapped by the Aux/IAA proteins. Purified IAA solution was collected and directly used to perform IAA determination by LC-MS/MS after elution. The gravity column filled with GST resin could be reused after regeneration.
S2-pIEx-3 System is Favourable for Expression of Higher Bioactivity Aux/IAA Proteins
The recombinant proteins derived from various expression systems displayed differing amounts of bioactivity, and the pIEx-3 was suitable for expressing highly bioactive proteins. The pIEx-3 vector was previously designed for the cloning and expression of proteins in transiently transfected Spodoptera-derived insect cells. Transcription is driven by the AcNPV-derived hr5 enhancer and the immediate early promoter IE1. pIEx-3 contains the coding sequence for the signal peptide of adipokinetic hormone (AKH) to allow the secretion of the expressed protein [48, 49]. pIEx-3 also contains the N-terminal GST-Tag, His-Tag, and S-Tag, as well as a C-terminal HSV-Tag coding sequence for protein detection and purification (Fig. 2a). More than 40 proteins, including cytoplasmic protein kinases or regions of a receptor with kinase activity, kinase interacting proteins, phospolipases, nuclear transport proteins, phosphatases, and heat shock proteins have been expressed using this InsectDirect System, and the yields were as high as 8 mg from 100 mL culture in Sf9 and Sf21 cells . In this study, the recombinant proteins displayed higher bioactivity, although lower recombinant protein yield was obtained in the stable transfectant S2 line (Fig. 4d). One potential reason for this could be that the post-translation, modification and folding in insects may differ from these processes in native plants.
The Aux/IAA Proteins Exhibited Different IAA Affinity
Aux/IAA proteins belong to a large protein family. In Arabidopsis, 29 Aux/IAA proteins have been identified, and the existence of multiple Aux/IAA–ARF combinations may mediate specific responses [51–54]. The type of Aux/IAA protein may be the key factor in the auxin trapping that occurs in plant cells, since different Aux/IAA–TIR1 co-receptors varied greatly in their affinity for auxin . In this study, the higher IAA affinity observed with the IAA7 protein confirms this hypothesis.
Affinity IAA Extraction and Purification Methods Showed Great Potential
The ideal extraction and purification method for a particular phytohormone must be simple, rapid and specific to reduce its degradation and improve its recovery, as plant scientists require accurate quantification in trace plant tissues. It is critical to keep developing new extraction and purification methods for these compounds to satisfy the need for greater precision that accompanies the highly active field of investigation into the mechanisms of phytohormone actions. Currently, organic extraction and solid-phase extraction are the most frequently used methods in phytohormone extraction and purification prior to their analysis by LC-MS/MS. MIPs and some developed operational strategies, such as two-dimensional HPLC, online 2D HPLC and high-performance thin-layer chromatography (HPTLC), have been employed to purify phytohormones [23, 55, 56].
To further simplify the extraction procedure and improve specificity for a particular phytohormone, the use of bioactive protein/complex applications shows great potential in phytohormone sample preparation. For example, some functional proteins in the signalling pathway of auxin, such as auxin binding proteins, receptors and co-receptors, are able to accurately recognize and bind auxin molecules even below the fM level in plant cells. Thus, Aux/IAA proteins display a high level of affinity and selectivity towards IAA. In this study, Aux/IAA proteins were used as a selective sorbent, and an affinity IAA extraction and purification method was developed. This method eliminated the problem of interference by structurally similar compounds and simplified the procedure of IAA extraction and purification from plant crude extracts. Our application of Aux/IAA protein validated the use of phytohormonal signalling proteins in phytohormone extraction and purification.
IAA can be extracted and purified from crude sample extracts using our method, but unfortunately not all of the protein was recovered. One reason for this could be the limited bioactivity and stability of the Aux/IAA proteins. To achieve highly stable Aux/IAA proteins, other expression systems should be tested to produce recombinant proteins; other tags should be used to solidify target proteins, and other buffers should be tested for the isolation and purification of proteins and the trapping of IAA. To achieve higher binding capabilities, the IAA co-receptor, which complexes with both the TIR1 and Aux/IAA protein, should be further tested as a sorbent since this co-receptor displayed a much greater affinity for auxin .
We expressed Aux/IAA proteins in E. coli and S2 cells and developed a method for IAA extraction and purification based on the recombinant proteins of Aux/IAAs. This method can be used for reference in bioactivity study and detection practice for other bioactive molecules.
Auxin Response Factor
AUXIN RESISTANT/INDOLE-3-ACETIC ACID INDUCIBLE
Dispersive Liquid–Liquid Microextraction
Double Layered SPE
Hollow Fiber-Based Liquid–Liquid–Liquid Microextraction
High pH Buffer
Low pH Buffer
Molecularly Imprinted Polymers
Polymer Monolith Microextraction
TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX
This research was carried out with funding from National Natural Science Foundation of China and Scientific Research Fund of Hunan Provincial Education Department. We also wish to thank Professor Keqin Peng and Professor Ruozhong Wang for their valuable advises.
This work was financially supported by National Natural Science Foundation of China (Grant No. 91317312, 31570372 and 9141730003) and Scientific Research Fund of Hunan Provincial Education Department (Grant No. 13 K065 and 15 K061).
Availability of Data and Materials
DNA sequences used in this study and cloning data were available as Additional file. Additional file 1: S1 Aux/IAA sequences cloned in this paper. Additional file 1: S2 IAA1 cloning and sequencing. Additional file 1: S3 IAA7 cloning and sequencing. Additional file 1: S4 IAA28 cloning and sequencing.
LX and YS designed the project, analyzed data and wrote the manuscript. YS, WL, XC, HL and YH conducted the experiments. WL analyzed the data and revised the manuscript. All authors review and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for Publication
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