CBM21 starch-binding domain: a new purification tag for recombinant protein engineering.

The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.

Role of the linker region in the expression of Rhizopus oryzae glucoamylase.

BACKGROUND: Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132-167) connects the two functional domains, but its structural and functional roles are unclear. RESULTS: To characterize the linker sequences of RoGA and its involvement in protein expression, a number of RoGA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of RoGA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains. CONCLUSION: Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of RoGA.

Abrogation of streptococcal pyrogenic exotoxin B-mediated suppression of phagocytosis in U937 cells by Cordyceps sinensis mycelium via production of cytokines.

Streptococcal pyrogenic exotoxin B (SPE B) is a virulent factor in group A streptococcal infection. We previously showed that SPE B reduced phagocytosis in human monocytic U937 cells. Here we show that the mycelium extract of Cordyceps sinensis (CS), a Chinese immunomodulatory herbal medicine, increased phagocytosis in U937 cells. Neither heat nor trypsin pretreatment prevented CS extract from causing this increase. Further studies indicated that SPE B-mediated suppression of U937 cell phagocytic activity was abrogated by CS extract. Factors in the conditioned medium from CS-extract-treated U937 cells were responsible for blocking the SPE B-mediated suppression of phagocytosis. Heating the conditioned medium eliminated the increase, which suggested that the U937-cell protein products augmented phagocytosis. Analyzing cytokine mRNA expression of U937 cells revealed increases in interferon-gamma (IFN-gamma), interleukin (IL)-12 p35 and p40, and tumor necrosis factor-alpha (TNF-alpha), but not in IL-1beta, IL-6, or IL-8. Treating U937 cells with anti-IFN-gamma, IL-12, and TNF-alpha antibodies also eliminated the conditioned medium-induced increase in phagocytosis. Taken together, SPE B inhibited phagocytosis, but CS mycelium extract abrogated this inhibition by causing cytokine production.

Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion.

We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.

Cordyceps sinensis mycelium protects mice from group A streptococcal infection.

Group A streptococcus (GAS) infection can cause severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Cordyceps sinensis, a Chinese herbal medicine, is an immunomodulator. In this study the air-pouch bacterial inoculation model was used to investigate the protective efficacy of C. sinensis mycelium extract against GAS infection. Force-feeding mice with C. sinensis mycelium extract for 3 consecutive days before GAS infection increased the survival rate and reduced local skin-tissue injury compared with mice fed PBS. Bacterial numbers in the air pouch exudates from C. sinensis-treated mice were lower than those from PBS-treated mice. Blood and organs in PBS-treated mice showed bacterial dissemination, but those in C. sinensis-treated mice did not. Three days of pretreatment with C. sinensis extract followed by C. sinensis treatment every other day after GAS infection resulted in 100% survival. The post-GAS-infection levels of aspartate aminotransferase, alanine aminotransferase and blood urea nitrogen in the sera of C. sinensis-treated mice were lower than those of PBS-treated mice. Taken together, these results show that C. sinensis mycelium extract protects by decreasing bacterial growth and dissemination, thereby increasing mouse survival rate. IL-12 and IFN-gamma expression and macrophage phagocytic activity also increased after C. sinensis treatment.

Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations.

Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris.

In vivo stimulatory effect of Cordyceps sinensis mycelium and its fractions on reproductive functions in male mouse.

Cordyceps sinensis (CS), an Ascomycetes fungus parasitic to Lepidoptera larvae, has been traditionally used as nutritious food for the enhancement on sexual performance and the restitution of impairment in sexual function in Chinese society. We have previously demonstrated the stimulatory effect of CS and its fractions on steroidogenesis both on primary mouse Leydig cells and MA-10 mouse Leydig tumor cells. In the present studies, we determined the in vivo effects of CS and its fractions on steroidogenesis in mouse. Different concentrations of CS and CS fractions (0.02 and 0.2 mg/g body weight) were fed to immature or mature mice from 1 to 7 days. The plasma levels of testosterone were evaluated by radioimmunoassay. The weights of reproductive organs were also determined. Results illustrated that CS significantly induced plasma testosterone levels both in immature and mature mice in 3 and/or 7 days treatment (p < 0.05). F2 and F3 at 0.02 and/or 0.2 mg/g body weight for different feeding duration could also significantly stimulated plasma testosterone levels both in immature and mature mice (p < 0.05). In general, CS, F2 and F3 didn't have considerable effect on the weights of reproductive organs. Taken together, these studies illustrate that CS and its fractions significantly stimulated in vivo mouse testosterone production.

Five new maleic and succinic acid derivatives from the mycelium of Antrodia camphorata and their cytotoxic effects on LLC tumor cell line.

Five new maleic and succinic acid derivatives were isolated from the mycelium of Antrodia camphorata. Their structures were determined by various spectroscopic means. Maleimide derivatives 2 and 3 showed appreciable cytotoxic activity against LLC cells.

In vivo and in vitro stimulatory effects of Cordyceps sinensis on testosterone production in mouse Leydig cells.

The in vivo and in vitro effects of Cordyceps sinensis (CS) and its extracted fractions on the secretion of testosterone in mice were studied. CS, F2 (water soluble protein), and F3 (poorly water soluble polysaccharide and protein) significantly stimulated in vitro testosterone production in purified mouse Leydig cells. However, F1 (water soluble polysaccharide) had no effect (p>0.05). F2 and F3 stimulated in vitro testosterone production in dose- and time-dependent relationships with maximal responses at 3 mg/ml for 3 h (p<0.05). An in vivo study illustrated that testosterone levels in plasma were significantly increased by CS, F2, and F3, respectively (p<0.05). Because CS, F2, and F3 stimulated both in vitro and in vivo testosterone secretions in mice, it is possible that CS might contribute to an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males.