Trx Tag Research Articles

Display of CCL21 on cancer cell membrane through genetic modification using a pH low insertion peptide

CCL21, a secondary lymphoid tissue chemokine, plays an important role in generating an effective anti-tumor immune response. In this study, a genetically modified CCL21 was developed by inserting a pH low insertion peptide to establish a CCL21-rich microenvironment for tumors. The fusion tag thioredoxin (Trx) was designed and fused at the N-terminal of the recombinant protein to protect it from being irrevocably misfolded in microbial host cells. The prokaryotic expression vector pET32a-CCL21-pHLIP was constructed and successfully expressed in E. coli BL21 (DE3) with a soluble expression form and a molecular weight of ~35 kDa. The induction conditions were optimized to obtain an extremely high yield of 6.7 mg target protein from 31.1 mg total protein. The 6xHis tagged Trx-CCL21-pHLIP was purified using Ni-NTA resin, and it was confirmed using SDS-PAGE and Western blot analyses. Consequently, the Trx-CCL21-pHLIP protein was successfully displayed on the cancer cell surface in a weak acidic microenvironment and showed the same ability as CCL21 in recruiting CCR7-positive cells. Additionally, the CCL21 fusion protein with or without Trx tag showed similar functions. Therefore, the study implies the feasibility of directing a modular genetic method for the development of protein-based drugs.

A simple and efficient method for cytoplasmic production of human enterokinase light chain in E. coli

Human enterokinase light chain (hEKL) cDNA sequence was designed with the help of codon optimization towards Escherichia coli codon preference and ribosome binding site design and artificially synthesized with a thioredoxin fusion tag at the N-terminal and a five his-tag peptide at the C-terminal. The synthetic hEKL gene was cloned into the pET-15 expression vector and transferred into the three different expression strains of E. coli BL21(DE3), NiCo21, and SHuffle T7 Express. Different growth and induction conditions were studied using a statistical response surface methodology (RSM). Recombinant hEKL protein was expressed at high levels in soluble form with 0.71 mM IPTG after 4 h of induction at 25 °C. Autocatalytic process cleaved TRX tag with enterokinase recognition site by the impure hEKL and yielded the mature enzyme. The target protein was then purified to homogeneity (> 95%) by affinity chromatography. The activity of hEKL was comparable to the commercial enzyme. From 1 L culture, 80 mg pure active hEKL was obtained with the specific activity of 6.25 × 102 U/mg. Three main parameters that help us to produce the enzyme in the folded and active form are the type of strain, SHuffle T7 strain, TRX and histidine fusion tags, and growth conditions including the increase of OD of induction and IPTG concentration and the decrease of induction temperature.

The expression, purification, and functional evaluation of the novel tumor suppressor fusion protein IL-24-CN.

Interleukin-24 (IL-24) can specifically induce apoptosis in a broad range of cancer cells without harming normal cells. The interaction of contortrostatin (CN) with integrins on angiogenic vascular endothelial and tumor cells is modulated by the RGD motifs that can significantly inhibit metastasis and angiogenesis. To achieve superior therapeutic efficacy by combining anti-metastasis with tumor-selective apoptosis activity, CN was fused at the C-terminus of IL-24 with a flexible linker (G4S)2, and the recombinant IL-24-CN was expressed in Escherichia coli as a Thioredoxin (Trx)/IL-24-CN fusion protein. The target protein was purified using nickel affinity chromatography. Furthermore, we simplified the purification process by purifying Trx-IL-24-CN and cleaving the Trx tag in one step. The final yield of IL-24-CN was 27.6mg/L based on flask fermentation. In vitro activity assay demonstrated that the recombinant IL-24-CN could more effectively suppress tumor growth and induce apoptosis of melanoma cells. Scratch and transwell assays suggested that IL-24-CN strongly reduced the migration and invasion behavior of melanoma cells. Immunofluorescence analysis and cell adhesion assay showed that CN could evidently improve the tumor inhibition capability of IL-24 by enhancing the affinity of recombinant protein toward cancer cells. In summary, a highly efficient strategy was developed for producing the bioactive IL-24-CN from prokaryotic cells, supporting IL-24-CN in melanoma therapy.Key points• Efficient heterologous production of recombinant IL-24-CN in E. coli using Trx fusion strategy.• Improved tumor growth suppression and apoptosis induction potency of IL-24-CN.• Enhanced cell adhesion ability of IL-24-CN in cancer cells.

Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Bacterial expression of 183-227aa region of HER3 extracellular domain I and preparation and identification of its polyclonal antibodies

To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in E.coli BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B. The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG. We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.

Thermostable Esterase estUT1 from Ureibacillus thermosphaericus: Effect of TrxA Tag on the Enzyme Properties

The thermostable esterase from the bacterium Ureibacillus thermosphaericus was expressed with Trx tag from plasmid pET32b-estUT1 under T7 promoter in E. coli BL21(DE3). The specific activity and relative thermal stability of the tagged enzyme increased from 45.2 to 65.8% (1 h at 70°С). The additional TrxA tag does not affect the pH optimum of enzyme activity and substrate specificity. At the same time, the absence of the TrxA tag resulted in a significant increase in the stability of estUT1 in during incubation with various chemicals, including ethanol and methanol. The maximum catalytic efficiency (kcat/KM) for esterase was observed in the absence of the TrxA tag and was 280.0 s−1 mM−1. Thereby fusion with TrxA tag promotes the enzyme secretion in the dissolved form, but reduces its thermal stability.

Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli

In this study, the cDNA of mature bovine interferon-α (BoIFN-α) gene was cloned and expressed in the Escherichia coli expression system. Total RNA was extracted from bovine liver cells and reverse transcribed into cDNA, and then BoIFN-α gene fragment was amplified with specific primers by PCR method before being ligated into the pET-32a (+) expression vector. Since the pET-32a (+) plasmid expresses the thioredoxin A (TrxA) tag protein, it can increase the solubility of the target protein. In order to express the target protein, the pET-32a-BoIFN-α recombinant plasmid was transformed into E. coli Rosetta (DE3) strain followed by IPTG induction. It was found that the recombinant fusion protein Trx-rBoIFN-α was finally expressed in a soluble form in the host cell, accounting for about 33.45% of the total cellular protein. The protein was purified by two-step chromatography, including Ni2+-chelating Sepharose affinity chromatography and DEAE anion exchange chromatography. The purity of the purified product reached 93.0%, and the cytopathic effects (CPE) inhibition method was adopted to determine the antiviral activity of the expressed fusion protein in vesicular stomatitis virus/ Madin-Darby bovine kidney cells titration system. The specific activity of the recombinant fusion protein Trx-rBoIFN-α was measured as (3.6 ± 0.25) × 106 U/mg. After cutting of the Trx tag protein by enterokinase digestion, the remaining solo protein was confirmed as rBoIFN-α protein by Western blot assay. These findings will enable us to produce a large number of bioactive rBoIFN-α protein for future application.

Drosophila CG1275 Expression, Antibody Production, and Western Blotting

Little is known about iron absorption in insects. One potential mechanism involves the action of a ferric reductase to reduce Fe(III) to Fe (II), which can enter the cell through a divalent metal transporter. CG1275 is a potential ferric reductase that is found in Drosophila melanogaster. By replicating, cloning, and expressing the N‐terminus of CG1275 we can develop a reagent to allow us to observe a protein knockdown after RNA interference (RNAi) experiments. We cloned a cDNA encoding the N‐terminal 107 amino acids of CG1275‐PA, the region before the first transmembrane helix, into pET‐32a. This was confirmed by restriction digestion. Expression of the N‐terminus of CG1275 along with a thioredoxin (Trx) tag and 6xHis tag from the vector was performed in Bl21 (DE3) E. coli. We then purified the protein using Nickel‐column affinity chromatography. The purified protein was separated by SDS‐PAGE and the protein bands of interest were cut out and sent for antibody production. The newly developed antibodies were then separated to remove antibodies that recognize the Trx tag. The antibodies to CG1275 were then used for western blots of S2 cell lysates to determine if they recognize CG1275 and are able to verify successful CG1275 knockdown after RNAi experiments.Support or Funding InformationNSF (IOS‐1656407)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.

Optimized Procedure for Recovering HIV-1 Protease (C-SA) from Inclusion Bodies.

HIV-1 is an infectious virus that causes acquired immunodeficiency syndrome (AIDS) and it isone of the major causes of deaths worldwide. The production of HIV-1 protease (PR) on alarge scale has been a problem forscientists due to its cytotoxicity, low yield, insolubility, and low activity. HIV-1C-SA protease has been cloned, expressed, and purified previously, however, with low recovery (0.25mg/L). Herein we report an optimal expression and solubilisation procedure to recover active HIV-1C-SA protease enzyme from inclusion bodies. The HIV protease was expressed in seven different vectors (pET11b, pET15b, pET28a pET32a, pET39b, pET41b and pGEX 6P-1). The highest expression was achieved when the vectorpET32a (Trx tag) was employed. A total of 19.5mg of fusion protein was refolded of which 5.5mg of active protease was obtained after cleavage. The free protease had a high specific activity of 2.81 µmoles/min/mg. Interestingly the Trx-fusion protein also showed activity closer (1.24 µmoles/min/mg) to that of the free protease suggesting that thepET32a vector (Trx tag) expressed in BL21(DE3) pLysS provides a more efficientway to obtain HIV-1 protease.

Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein.

Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated. Moreover, the effect of temperature and isopropyl-β-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), Rosetta™ (DE3), and Origami™ (DE3). The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in Rosetta™ (DE3) and Origami™ (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system.

Cloning and Heterologous Expression of Plantaricin ZJ5, a Novel Bacteriocin from Lactobacillus plantarum ZJ5, in Escherichia coli

A novel bacteriocin, plantaricin ZJ5 (PZJ5) was yielded from Lactobacillus plantarum ZJ5, cloned, and produced in Escherichia coli BL21 (DE3) pLys. The PZJ5 structural gene was fused with a Trx tag, and cloned into the pET32a plasmid under the control of the inducible lac operon. Induction was performed with isopropyl-β-D-thiogalactopyranoside (IPTG), with subsequent overexpression of the fusion protein, followed by purification to homogeneity via His affinity chromatography. Recombinant E. coli produced greater quantities of PZJ5 than L. plantarum ZJ5, and PZJ5 in E. coli was expressed in the form of soluble material. Biologically active PZJ5 was recovered by cleaving the purified fusion protein using enterokinase. The released PZJ5 demonstrated antibacterial activity against Micrococcus luteus. In this study, an inexpensive biological method using a Trx fusion system was presented, and for the first time, bacteriocin PZJ5 was expressed and purified in E. coli.

Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli

Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl β-d-1-thiogalactopyranoside, and it was purified by Ni2+-nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests.

Expression and Purification of Soluble form of Human Parathyroid Hormone (rhPTH1-34) by Trx Tag in E. coli

Expression and Purification of Soluble form of Human Parathyroid Hormone (rhPTH1-34) by Trx Tag in E. coli

Production of tropane alkaloids by biotransformation using recombinant Escherichia coli whole cells

Tropane alkaloids, such as hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine, are secondary metabolites that were traditionally applied in medicine due to their anticholinergic activity. Hyoscyamine is converted into 6β-hydroxyhyoscyamine and scopolamine by Hyoscyamine-6β-hydroxylase (H6H). Nowadays, these bioactive compounds are obtained from natural producer plants due to the cost and complexity of their chemical synthesis. In the present work we explored the development of an alternative strategy for the production of the most valuable alkaloids, 6β-hydroxyhyoscyamine and scopolamine, using Escherichia coli harboring the H6H enzyme as biocatalysts. In addition, the protein extracts of the induced bacteria were assayed for the transformation of hyoscyamine into the more valuable alkaloids. For this purpose the h6hcDNA, previously amplified from Brugmansia candida total RNA preparations, was inserted in frame to the trx tag into the pET32a(+) vector. E. coli Origami strains were used as host for the expression. The strategy allowed us to produce enough quantities of a soluble and functional enzyme. Protein extracts and whole cells of the induced bacteria were able to transform hyoscyamine into the valuable products. In addition, we found that except from 2-oxoglutarate, no supplementation of the reaction mixture with the cofactors and co-substrates was needed. The process developed in this work is attractive since it could become an alternative to the traditional isolation of 6β-hydroxyhyoscyamine and scopolamine.

Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein.

Background:Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein.Materials and Methods:In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains.Results:Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins.Conclusions:The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains.

Generation of scFv specific to human VEGFR-3 from the neutralizing mAb BDD073.

In our previous study, we have produced a neutralizing mAb of vascular endothelial growth factor receptor 3 (VEGFR-3), specifically BDD073, which could inhibit angiogenesis in the CAM model. However, the clinical application of BDD073 is restricted due to its mouse origin, which might cause human anti-mouse antibody reactions. Herein, we generated functional recombinant single-chain variable fragments (scFv) from mAb BDD073 producing mouse hybridoma cells. The scFv gene containing variable regions of heavy and light chains of BDD073 was cloned into an expression vector with trx tag and expressed in Escherichia coli BL21 (DE3). The recombinant scFv was purified and refolded with Ni-NTA agarose metal affinity column. The bacterially expressed scFv showed moderate potency and specificity to the human VEGFR-3. It may serve as a potential candidate of anti-VEGFR3 treatment for biotechnological and therapeutic applications.

High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli.

Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000U/L and 1,278U/L/H in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650U/L and 1,228U/L/H, respectively, by scaling up SmL production in a 7.0L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs.

Expression and Purification of Serine Protease Inhibitor, OH-TCI, in <i>Escherichia coli</i> as a Thioredoxin Fusion Protein

The OH-TCI was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom with strong inhibitory activity against trypsin and chymotrypsin. Thus it is theoretically attractive in ameliorating the effects of acute pancreatitis and reducing allogeneic blood product transfusion during cardiac surgery. To accomplish preclinical evaluation with OH-TCI, large-scale production of OH-TCI is produced in Escherichia coli. The optimized OH-TCI codons were cloned into pET32a (+). OH-TCI expressed as Trx tag is solubility as fusion bodies. The amount of the purified Trx-OH-TCI from 1 liter culture was roughly 200 mg. The inhibitor constants (Ki) of Trx-OH-TCI, similar to the native OH-TCI, were 2.67×10-7 and 3.60×10-7 M M for trypsin and chymotrypsin, respectively.

Cloning, prokaryotic expression, and bioactivity of the calmodulin gene ofMagnaporthe grisea

The cDNA encoding the calmodulin (CaM) protein was isolated from the mycelia of Magnaporthe grisea PO-041 strain by reverse transcriptase (RT)-PCR and named MgCaM. High similarities in sequence to other reported CaMs suggested that CaM is highly conserved. The MgCaM gene was expressed in Escherichia coli BL21 using a pET32a(+) plasmid. MgCaM with or without a Trx tag exhibited a Ca(2+)-dependent electrophoretic migration shift, suggesting that the activity of MgCaM depends on the presence of Ca(2+). Western blot analysis showed that Trx-MgCaM possessed specific immunoreactivities and could be specifically recognized by an anti-AtCaM2-Ig polyclonal antibody raised against AtCaM2 of Arabidopsis. Immunocytolocalization of MgCaM indicated that the number of gold particles was the most in germ tubes, next to conidia, followed by appressoria and the mycelia from liquid media, and the least in the aerial hypha from solid media. Also, in the germ tube, mycelium from liquid media and appressorium, the density of gold particles in the cell wall was markedly higher than that in the cytoplasm. In conidia, the number of MgCaM particles in the cytoplasm was obviously higher than that in the cell wall. However, in the aerial hypha, MgCaM only distributed at the cytoplasm and no gold particle was detected in the cell wall.