PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.
Animals ; Antiviral Agents ; Humans ; Interferon alpha-2 ; metabolism ; Interferon-alpha ; biosynthesis ; pharmacokinetics ; Polyethylene Glycols ; pharmacokinetics ; Protein Structure, Secondary ; Rats ; Recombinant Proteins ; biosynthesis ; pharmacokinetics ; pharmacology ; Reproducibility of Results ; Transglutaminases ; metabolism

Direct secretory expression of active microbial transglutaminase (MTG) using heterologous hosts is a promising strategy, although its production level still needs to be improved for industrial production. Pichia pastoris is one of the most efficient expression systems developed in recent years. In this study, secretory expression of active MTG was successfully achieved by co-expressing the pro sequence and mature MTG genes in P. pastoris. Furthermore, we optimized the copy number of pro/MTG expression cassettes and the fermentation conditions. MTG production level reached 7.3 U/mL in 1-liter fermentor through high density fermentation, providing the feasiblity for industrial scale preparation of MTG.
Fermentation ; Genetic Vectors ; genetics ; Pichia ; enzymology ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Streptomyces ; enzymology ; Transglutaminases ; biosynthesis ; genetics

Transglutaminase 2 (TG2), a cross-linking enzyme, is involved in drug resistance and in the constitutive activation of nuclear factor kappa B (NF-kappaB). We investigated the association of non-small cell lung cancer (NSCLC) treatment efficacy with TG2 and NF-kappaB expression in 120 patients: 102 with adenocarcinoma and 18 with other histologic types. All patients underwent surgery; 88 received adjuvant chemotherapy, with 28 receiving platinum-based doublet chemotherapy as first-line treatment and 29 receiving epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Patients' TG2 and NF-kappaB expression values were calculated semiquantitatively. The median TG2 value was 50 (range, 0-300) and the median NF-kappaB value was 20 (range, 0-240). Disease-free survival did not differ between the low- and high-TG2 groups. Among patients who received palliative platinum-based doublet chemotherapy, progression free survival (PFS) was longer in the low-TG2 group than in the high-TG2 group (11.0 vs. 7.0 months; P=0.330). Among those who received EGFR-TKI therapy, PFS was also longer in the low-TG2 group than in the high-TG 2 group (11.0 vs. 2.0 months; P=0.013). Similarly, in EGFR wild-type patients treated with EGFR-TKI, PFS was longer in patients with low TG2 expression (9.0 vs. 2.0 months; P=0.013). TG2 expression levels can predict PFS in patients with NSCLC treated with EGFR-TKI.
Adenocarcinoma/*drug therapy/mortality/surgery ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents/therapeutic use ; Carcinoma, Non-Small-Cell Lung/*drug therapy/mortality/surgery ; Disease-Free Survival ; Female ; GTP-Binding Proteins/*biosynthesis ; Humans ; Lung Neoplasms/*drug therapy/mortality/surgery ; Male ; Middle Aged ; NF-kappa B/biosynthesis ; Protein Kinase Inhibitors/therapeutic use ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/genetics ; Transglutaminases/*biosynthesis ; Treatment Outcome

Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.
Animals ; Animals, Newborn ; Bordetella bronchiseptica ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mice ; Neutralization Tests ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transglutaminases ; biosynthesis ; genetics ; Virulence Factors, Bordetella ; biosynthesis ; genetics

Microbial transglutaminase, which could catalyze the cross-linking of many proteins or non-protein materials, has been widely used in food, pharmaceutical and textile industry. To enhance the yield of the enzyme and establish corresponding platform for molecular modification, the researchers of Japanese Ajinomoto began to construct the recombinant strain producing transglutaminase in the 1990s. So far, the enzyme has been successfully expressed in different expression systems. Some of the recombinant strains are more productive than wild strains. Recently, progress has been made in the molecular modification of microbial transglutaminase, and the activity, thermo-stability and specificity of the enzyme are improved. This review briefly summarized and analyzed the strategies involved in these studies, and noted its trends.
Bacterial Proteins ; biosynthesis ; genetics ; Catalysis ; Enzyme Stability ; Mutagenesis, Site-Directed ; Mutant Proteins ; genetics ; metabolism ; Protein Engineering ; methods ; Streptomyces ; enzymology ; Substrate Specificity ; Transglutaminases ; biosynthesis ; genetics

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON and tTG-ASDON2 were significantly decreased as compared with that of the controls (P < 0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.
Cells, Cultured ; Glaucoma, Open-Angle/metabolism ; Oligonucleotides, Antisense/*pharmacology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Trabecular Meshwork/cytology ; Trabecular Meshwork/*metabolism ; Transglutaminases/*biosynthesis ; Transglutaminases/genetics ; Transglutaminases/*pharmacology

To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.
Animals ; Cattle ; Cells, Cultured ; Glaucoma, Open-Angle ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trabecular Meshwork ; metabolism ; Transglutaminases ; biosynthesis ; genetics

To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.
Cells, Cultured ; Glaucoma, Open-Angle/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trabecular Meshwork/*metabolism ; Transglutaminases/*biosynthesis ; Transglutaminases/genetics

OBJECTIVECalcium Glucarate (Cag), Ca salt of D-glucaric acid is a naturally occurring non-toxic compound present in fruits, vegetables and seeds of some plants, and suppress tumor growth in different models. Due to lack of knowledge about its mode of action its uses are limited in cancer chemotherapy thus the objective of the study was to study the mechanism of action of Cag on mouse skin tumorigenesis.

METHODSWe have estimated effect of Cag on DMBA induced mouse skin tumor development following complete carcinogenesis protocol. We measured, epidermal transglutaminase activity (TG), a marker of cell differentiation after DMBA and/or Cag treatment and [3H] thymidine incorporation into DNA as a marker for cell proliferation.

RESULTSTopical application of Cag suppressed the DMBA induced mouse skin tumor development. Topical application of Cag significantly modifies the critical events of proliferation and differentiation TG activity was found to be reduced after DMBA treatment. Reduction of the TG activity was dependent on the dose of DMBA and duration of DMBA exposure. Topical application of Cag significantly alleviated DMBA induced inhibition of TG. DMBA also caused stimulation of DNA synthesis in epidermis, which was inhibited by Cag.

CONCLUSIONCag inhibits DMBA induced mouse skin tumor development. Since stimulation of DNA synthesis reflects proliferation and induction of TG represents differentiation, the antitumorigenic effect of Cag is considered to be possibly due to stimulation of differentiation and suppression of proliferation.

9,10-Dimethyl-1,2-benzanthracene ; toxicity ; Administration, Topical ; Animals ; Anticarcinogenic Agents ; therapeutic use ; Carcinogens ; toxicity ; Cell Division ; drug effects ; DNA ; biosynthesis ; Enzyme Inhibitors ; toxicity ; Female ; Glucaric Acid ; therapeutic use ; Mice ; Skin Neoplasms ; chemically induced ; enzymology ; prevention & control ; Thymidine ; metabolism ; Transglutaminases ; metabolism

OBJECTIVETo investigate expression of tissue transglutaminase (tTG) protein and its role in carcinogenesis of brain tumors.

METHODStTG protein was detected by immunohistochemical method in 62 astrocytomas, 18 oligodendrogliomas, 30 benign meningiomas, 30 pituitary adenomas and 10 normal brain tissues.

RESULTS(1) In brain tumors, tTG protein expression was heterogeneous locating in tumor and endothelial cells. (2) Immunoreactivity of tTG protein was significantly different between different grades of astrocytomas. (3) Expression intensity of tTG protein in glioma was higher than that in benign brain tumors. (4) Strong expression of tTG protein in tumor cell was obtained around the necrosis foci and apoptotic cells in astrocytomas.

CONCLUSIONStTG protein expression contributed to tumor malignant progression in malignant brain tumors.

Adolescent ; Adult ; Aged ; Apoptosis ; Astrocytoma ; enzymology ; pathology ; Brain Neoplasms ; enzymology ; pathology ; Child ; Endothelial Cells ; enzymology ; Female ; GTP-Binding Proteins ; biosynthesis ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Oligodendroglioma ; enzymology ; pathology ; Transglutaminases ; biosynthesis