Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Discovery of TCP-(MP)-caffeic acid analogs as a new class of agents for treatment of osteoclastic bone loss.

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.

A short guide on blue fluorescent proteins: limits and perspectives.

The advent of the so-called colorful biology era is in line with the discovery of fluorescent proteins (FPs), which can be widely used to detect the intracellular locations of macromolecules or to determine the abundance of metabolites in organelles. The application of multiple FPs that emit different spectra and colors could be implemented to precisely evaluate cellular events. FPs were initially established with the emergence of the green fluorescent protein (GFP) from jellyfish. Red fluorescent proteins (RFPs) from marine anemones and several corals adopt fluorescent chromophores that are similar to GFP. Chromophores of GFP and GFP-like FPs are formed through the oxidative rearrangement of three chromophore-forming residues, thereby limiting their application to only oxidative environments. Alternatively, some proteins can be fluorescent upon their interaction with cellular prosthetic cofactors and, thus, work in aerobic and anaerobic conditions. The modification of an NADPH-dependent blue fluorescent protein (BFP) also expanded its application to the quantization of NADPH in the cellular environment. However, cofactor-dependent BFPs have an intrinsic weakness of poor photostability with a high fluorescent background. This review explores GFP-derived and NADPH-dependent BFPs with a focus on NADPH-dependent BFPs, which might be technically feasible in the near future upon coupling with two-photon fluorescence microscopy or nucleic acid-mimickers. KEY POINTS: • Oxidation-dependent GFP-like BFPs and redox-free NADPH-dependent BFPs • GFPs of weak photostability and intensity with a high fluorescent background • Real-time imaging using mBFP under two-photon fluorescence microscopy.

One-step metal affinity purification of recombinant hFGF19 without using tags.

Human fibroblast growth factor 19 (hFGF19) belongs to the endocrine FGF19 superfamily and is considered a potential agent to treat severe or relapsing nonalcoholic fatty liver disease. Numerous studies have confirmed the beneficial effects of this hormone on the related symptoms of the disease and attempts at producing recombinant proteins in various hosts are steadily proliferating. Recently, we reported that authentic hFGF19 can be solubly expressed through combining synonymous codon substitutions and co-expression with disulfide-bond isomerase (DsbC) in Escherichia coli. However, during purification, hFGF19 without the His-tag occasionally co-eluted with His-tagged DsbC when using metal affinity chromatography, thereby requiring auxiliary purification steps to achieve apparent homogeneity. This phenomenon provides evidence that hFGF19 specifically interacts with immobilized Ni2+, which can thus be used as an alternative tool for the purification of hFGF19. Consequently, we could simply and reproducibly purify hFGF19 from cell lysates by using Ni2+-immobilized metal affinity chromatography and stepwise gradient elution with imidazole.

Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration.

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP+ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.

A new, reliable, and high-throughput strategy to screen bacteria for antagonistic activity against Staphylococcus aureus.

BACKGROUND: Antibiotic-resistant Staphylococcus aureus clones have emerged globally over the last few decades. Probiotics have been actively studied as an alternative to antibiotics to prevent and treat S. aureus infections, but identifying new probiotic bacteria, that have antagonistic activity against S. aureus, is difficult since traditional screening strategies are time-consuming and expensive. Here, we describe a new plasmid-based method which uses highly stable plasmids to screen bacteria with antagonistic activity against S. aureus. RESULTS: We have created two recombinant plasmids (pQS1 and pQS3) which carry either gfpbk or mCherry under the control of a S. aureus quorum-sensing (QS) promoter (agrP3). Using this recombinant plasmid pair, we tested 81 bacteria isolated from Holstein dairy milk to identify bacteria that had growth-inhibiting activity against S. aureus and suggest potential explanations for the growth inhibition. The stability test illustrated that pQS1 and pQS3 remained highly stable for at least 24 h in batch culture conditions without selection pressure from antibiotics. This allowed co-culturing of S. aureus with other bacteria. Using the newly developed pQS plasmids, we found commensal bacteria, isolated from raw bovine milk, which had growth-inhibiting activity (n = 13) and quorum-quenching (QQ) activity (n = 13) towards both S. aureus Sa25 (CC97) and Sa27 (CC151). The pQS-based method is efficient and effective for simultaneously screening growth-inhibiting and QQ bacteria against S. aureus on agar media. CONCLUSIONS: It was shown that growth-inhibiting and QQ activity toward pQS plasmid transformants of S. aureus can be simultaneously monitored by observing the zone of growth inhibition and reporter protein inhibition on agar plates. Newly identified antagonistic bacteria and their functional biomolecules are promising candidates for future development of probiotic drugs and prophylactics/therapeutics for bacterial infections including S. aureus. Furthermore, this new approach can be a useful method to find bacteria that can be used to prevent and treat S. aureus infections in both humans and animals.

Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA.

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic-hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

Dual Recognition of Sialic Acid and αGal Epitopes by the VP8* Domains of the Bovine Rotavirus G6P[5] WC3 and of Its Mono-reassortant G4P[5] RotaTeq Vaccine Strains.

Group A rotaviruses, an important cause of severe diarrhea in children and young animals, initiate infection via interactions of the VP8* domain of the VP4 spike protein with cell surface sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is also used in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for the VP8* domain of WC3 and its reassortant strains have not yet been identified. In the present study, HBGA- and saliva-binding assays showed that both G6P[5] WC3 and mono-reassortant G4P[5] strains recognized the αGal HBGA. The infectivity of both P[5]-bearing strains was significantly reduced in αGal-free MA-104 cells by pretreatment with a broadly specific neuraminidase or by coincubation with the α2,6-linked SA-specific Sambucus nigra lectin, but not by the α2,3-linked specific sialidase or by Maackia amurensis lectin. Free NeuAc and the αGal trisaccharide also prevented the infectivity of both strains. This indicated that both P[5]-bearing strains utilize α2,6-linked SA as a ligand on MA104 cells. However, the two strains replicated in differentiated bovine small intestinal enteroids and in their human counterparts that lack α2,6-linked SA or αGal HBGA, suggesting that additional or alternative receptors such as integrins, hsp70, and tight-junction proteins bound directly to the VP5* domain can be used by the P[5]-bearing strains to initiate the infection of human cells. In addition, these data also suggested that P[5]-bearing strains have potential for cross-species transmission.IMPORTANCE Group A rotaviruses initiate infection through the binding of the VP8* domain of the VP4 protein to sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is used as the backbone in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for their P[5] VP8* domain has remained elusive. Using a variety of approaches, we demonstrated that the WC3 and bovine-human mono-reassortant G4P[5] vaccine strains recognize both α2,6-linked SA and αGal HBGA as ligands. Neither ligand is expressed on human small intestinal epithelial cells, explaining the absence of natural human infection by P[5]-bearing strains. However, we observed that the P[5]-bearing WC3 and G4P[5] RotaTeq vaccine strains could still infect human intestinal epithelial cells. Thus, the four P[5] RotaTeq vaccine strains potentially binding to additional alternative receptors may be efficient and effective in providing protection against severe rotavirus disease in human.

Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5'-coding region in Escherichia coli.

OBJECTIVE: To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed. RESULTS: We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells. CONCLUSION: Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).

Conditional constitutive expression system of a drug protein in vivo by positive feedback loop using an inducer-independent artificial transcription factor.

Bacterial-mediated drug delivery is a potential and promising strategy for the specific treatment of cancer with therapeutic molecules, especially with genetically encoded proteins. These proteins must be tightly regulated due to cytotoxicity and thus are usually expressed under the control of the PBAD and TetA/TetR promoters in vivo. Since protein expression from these systems is triggered by exogenous inducer, periodic intravenous injection of inducer is necessary. However, these treatments can result in non-homogenous and/or inefficient expression of therapeutic proteins in vivo due to impeded diffusion and dilution of the inducer further from the injection site. To overcome these hurdles, we designed a conditional constitutive expression system equipped with the artificial transcription factor, AraCC, which has two operator-binding domains and simultaneously binds to the I1 and I2 operators of the PBAD promoter for gene expression in an arabinose-independent manner. Using this construct and the wild type protein AraC under the control of the PBAD promoter, we constructed a self-positive feedback system to constitutively express the therapeutic protein when the induction of AraC was triggered once using arabinose. This expression system could be useful in various cancer treatment strategies using bacteria to deliver genetically encoded drugs in vivo.

Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region.

Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.

Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in Escherichia coli.

Specific tags with defined amino acid residues are widely used to purify or probe target proteins. Interestingly, the tagging system occasionally results in an increase of the recombinant protein expression in vivo. Here, we systematically examined this phenomenon using a poly-histidine (His)-tag fused to N- or C-terminal region of green, red, and blue fluorescent proteins by quantification and uneven distribution in cytoplasm of Escherichia coli. This effect was further supported by the distinct over-expression of several unrelated proteins, such as esterase, neopullulanase, and chloramphenicol acetyltransferase, tagging with the same tag. These results suggest that a poly-His-tag placed at N-terminal region can induce over-expression of recombinant protein via subcellular uneven distribution in vivo.

A Method for Cell Culture and Maintenance of Ammonia-Oxidizing Archaea in Agar Stab.

Ammonia oxidizing archaea (AOA) are predominantly found and closely linked with geochemical cycling of nitrogen in non-extreme habitats. However, these strains have mainly been investigated using liquid cultures of enriched cells. Here, we provide an agar stab as a simple and reliable means of cultivating and maintaining AOA.

Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para-Azido-L-Phenylalanine.

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para-azido-L-phenylalanine (pAzF) in Escherichia coli. First, we expressed the maltose-binding protein (MBP)- fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation.

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

Gamma irradiation-engineered macrophage-derived exosomes as potential immunomodulatory therapeutic agents.

The modulation of macrophage polarization is a promising strategy for maintaining homeostasis and improving innate and adaptive immunity. Low-dose ionizing radiation has been implicated in macrophage immunomodulatory responses. However, studies on the relationship between exosomes and regulation of macrophage polarization induced by ionizing radiation are limited. Therefore, this study investigated the alterations in macrophages and exosomes induced by gamma irradiation and elucidated the underlying mechanisms. We used the mouse macrophage cell line RAW 264.7 to generate macrophages and performed western blot, quantitative reverse transcription-PCR, and gene ontology analyses to elucidate the molecular profiles of macrophage-derived exosomes under varying treatment conditions, including 10 Gy gamma irradiation. Exosomes isolated from gamma-irradiated M1 macrophages exhibited an enhanced M1 phenotype. Irradiation induced the activation of NF-κB and NLRP3 signaling in M1 macrophages, thereby promoting the expression of pro-inflammatory cytokines. Cytokine expression was also upregulated in gamma-irradiated M1 macrophage-released exosomes. Therefore, gamma irradiation has a remarkable effect on the immunomodulatory mechanisms and cytokine profiles of gamma-irradiated M1 macrophage-derived exosomes, and represents a potential immunotherapeutic modality.

Promising properties of cytochrome P450 BM3 reconstituted from separate domains by split intein.

Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes.

Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

PURPOSE: A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. PROCEDURES: We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. RESULTS: Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. CONCLUSIONS: We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

Construction of non-invasively constitutive expression vectors using a metagenome-derived promoter for soluble expression of proteins.

Expression of soluble and functional proteins has been one of the critical challenges to many aspects of synthetic biology, metabolic and protein engineering. Among the current methods for expression of target proteins, constitutive expression systems offer several advantages over inducible systems, which require a chemical or physical inducer. In a previous study, a G196 DNA fragment containing constitutive promoters was mined from the soil metagenome and evaluated for the expression of target proteins in the functional and soluble state. In this study, we further improved this system by constructing a series of constitutive expression vectors, pCEM (using the CEM promoter trimmed from G196), pCEMT (incorporating rrnB T1 and T2 terminator into the downstream region of MCS in pCEM) and pRCEMT (grafting the cis-acting region of pCEMT into a low-copy-number plasmid). Subsequently, genes encoding GFPuv, esterase 1767 and ß-glucosidase were subcloned into the resulting vectors, and their expression level and solubility were compared with those of IPTG-inducible vector systems pQE30 and pTrc99A. The extent of homogeneity and the ratio of the soluble fraction in the pRCEMT vector were relatively higher, without any delay of growth rate, than that of the pQE30 or pTrc99A. These results indicate that new expression vectors with moderate constitutive function could more easily lead to a homogenous population of cells expressing target proteins than those with conventionally inducible promoters.

Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay.

An efficient method for Pichia cell disruption that employs an aminopropyl magnesium phyllosilicate (AMP) clay-assisted glass beads mill is presented. AMP clay is functionalized nanocomposite resembling the talc parent structure Si8Mg6O20(OH)4 that has been proven to permeate the bacterial membrane and cause cell lysis. The recombinant capsid protein of cowpea chlorotic mottle virus (CCMV) expressed in Pichia pastoris GS115 was used as demonstration system for their ability of self-assembly into icosahedral virus-like particles (VLPs). The total protein concentration reached 4.24 mg/ml after 4 min treatment by glass beads mill combined with 0.2 % AMP clay, which was 11.2 % higher compared to glass beads mill only and the time was half shortened. The stability of purified CCMV VLPs illustrated AMP clay had no influence on virus assembly process. Considering the tiny amount added and simple approach of AMP clay, it could be a reliable method for yeast cell disruption.