Development of clone with novel TrpE fusion tag in E. coli for overexpression of trypsin in a bench-scale bioreactor.

Trypsin is a key enzyme under the serine proteases that is found in the pancreas which plays a key role in protein digestion. It cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. This enzyme has received greater attention mainly due to its increased use in the removal of fusion tags during protein purification and its role in the processing of biosimilars like insulin. The present study was carried out to develop a clone with Novel TrpLE1413(TrpE) Fusion Tag for enhanced expression of trypsin which helps in cost reduction of biosimilar processing. In our experiment we have used a synthetic bovine trypsin gene containing a novel fusion tag TrpE at its N terminus, which was cloned into the pET41b (+) vector and expressed in E. coli BL21 (DE3) in a lab-scale bioreactor. Using the optimized fermentation process with TrpE Fusion Tag, 27.8 g/L inclusion bodies were produced at the end of fermentation, of which 209 mg/L of active trypsin was obtained after purification. In contrast, previous reports have claimed to produce a maximum of 60 mg/L of the enzyme without the fusion tag. Thus based on our findings, the small size (less than 2 kDa) of TrpE tag and its hydrophobicity may reduce the loss incurred during the purification process. Hence, it could be discerned that the use of the TrpE fusion tag along with a robust fermentation process led to 3- 4 fold higher yield making it a commercially viable process facilitating an improved recovery of the enzyme.

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays.

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor.

BACKGROUND: Trypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate. RESULTS: The tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL-1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg-1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL-1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. CONCLUSIONS: SGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

High-level expression of PRSS3 correlates with metastasis and poor prognosis in patients with gastric cancer.

BACKGROUND AND OBJECTIVES: Serine protease-3 (PRSS3) is a known contributor to the genesis and development of malignant tumors, although its role in gastric cancer (GC) is still unclear. METHODS: PRSS3 expression in GC tissue samples and its relationship with clinicopathological features were analyzed. Effects of GC cellular responses to the introduction of small interfering RNA (siRNA)-mediated and short hairpin RNA (shRNA)-mediated interference with tumor PRSS3 expression were also assessed. RESULTS: PRSS3 was significantly upregulated in GC tissues, and PRSS3 protein levels were higher in tumors that developed metastases soon after the surgery compared with those that remained metastasis-free. High expression of PRSS3 was associated with tumor N staging and independently predictive of postoperative prognosis in patients with GC. The V1 variant of PRSS3 was primarily detected in GC tissue and cell lines, the others (V2-V4) being scarcely detectable. Methylation and demethylation drugs had no impact on expression levels of any PRSS3 transcriptional variant. The downregulated PRSS3 expression suppressed GC cell growth, migration, and invasion in vitro and in vivo. CONCLUSIONS: PRSS3 appears to act as an oncogene of GC. High PRSS3 expression portends postoperative metastasis, serving as an effective biomarker of poor therapeutic outcomes.

Rhododenol Activates Melanocytes and Induces Morphological Alteration at Sub-Cytotoxic Levels.

Rhododenol (RD), a whitening cosmetic ingredient, was withdrawn from the market due to RD-induced leukoderma (RIL). While many attempts have been made to clarify the mechanism underlying RIL, RIL has not been fully understood yet. Indeed, affected subjects showed uneven skin pigmentation, but the features are different from vitiligo, a skin hypopigmentary disorder, alluding to events more complex than simple melanocyte cytotoxicity. Here, we discovered that rhododenol treatment reduced the number of melanocytes in a pigmented 3D human skin model, Melanoderm™, confirming the melanocyte toxicity of RD. Of note, melanocytes that survived in the RD treated tissues exhibited altered morphology, such as extended dendrites and increased cell sizes. Consistently with this, sub-cytotoxic level of RD increased cell size and elongated dendrites in B16 melanoma cells. Morphological changes of B16 cells were further confirmed in the immunocytochemistry of treated cells for actin and tubulin. Even more provoking, RD up-regulated the expression of tyrosinase and TRP1 in the survived B16 cells. Evaluation of mRNA expression of cytoskeletal proteins suggests that RD altered the cytoskeletal dynamic favoring cell size expansion and melanosome maturation. Collectively, these results suggest that RD not only induces cytotoxicity in melanocytes but also can lead to a profound perturbation of melanocyte integrity even at sub-cytotoxic levels.

IL-1ß is a key cytokine that induces trypsin upregulation in the influenza virus-cytokine-trypsin cycle.

Severe influenza is characterized by a cytokine storm, and the influenza virus-cytokine-trypsin cycle is one of the important mechanisms of viral multiplication and multiple organ failure. The aim of this study was to define the key cytokine(s) responsible for trypsin upregulation. Mice were infected with influenza virus strain A/Puerto Rico/8/34 (H1N1) or treated individually or with a combination of interleukin-1ß, interleukin-6, and tumor necrosis factor α. The levels of these cytokines and trypsin in the lungs were monitored. The neutralizing effects of anti-IL-1ß antibodies on cytokine and trypsin expression in human A549 cells and lung inflammation in the infected mice were examined. Infection induced interleukin-1ß, interleukin-6, tumor necrosis factor α, and ectopic trypsin in mouse lungs in a dose- and time-dependent manner. Intraperitoneal administration of interleukin-1ß combined with other cytokines tended to upregulate trypsin and cytokine expression in the lungs, but the combination without interleukin-1ß did not induce trypsin. In contrast, incubation of A549 cells with interleukin-1ß alone induced both cytokines and trypsin, and anti-interleukin-1ß antibody treatment abrogated these effects. Administration of the antibody in the infected mice reduced lung inflammation area. These findings suggest that IL-1ß plays a key role in trypsin upregulation and has a pathological role in multiple organ failure.

A genomic region transcribed into a long noncoding RNA interacts with the Prss42/Tessp-2 promoter in spermatocytes during mouse spermatogenesis, and its flanking sequences can function as enhancers.

Spermatogenesis is regulated by many meiotic stage-specific genes, but how they coordinate the many individual processes is not fully understood. The Prss/Tessp gene cluster is located on mouse chromosome 9F2-F3, and the three genes at this site (Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4) are specifically activated during meiosis in pachytene spermatocytes. We searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus to elucidate how they are activated. We found eight DNase I HSs, three of which were testis germ cell-specific at or close to the Prss42/Tessp-2 promoter, and a testis-specific lncRNA, lncRNA-HSVIII, that was transcribed from a region adjacent to the Prss42/Tessp-2 gene. lncRNA-HSVIII transcripts localized to nuclei of most pachytene spermatocytes and the cytosol of stage-X pachytene spermatocytes and spermatids. Chromosome conformation capture revealed that the lncRNA-HSVIII locus specifically interacted with the Prss42/Tessp-2 promoter in primary and secondary spermatocytes. A 5.8-kb genome sequence, encompassing the entire lncRNA-HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp-2 promoter activity using a reporter-gene assay, yet this construct did not change lncRNA-HSVIII expression, indicating that the elevated promoter activity was likely through enhancer activity. Indeed, both upstream and downstream regions of the lncRNA-HSVIII sequence significantly increased Prss42/Tessp-2 promoter activity. Our data therefore identified the direct interaction of a genomic region in the lncRNA-HSVIII locus with the Prss42/Tessp-2 promoter in spermatocytes, and suggested that sequences adjacent to the lncRNA function as enhancers for the Prss42/Tessp-2 gene. Mol. Reprod. Dev. 83: 541-557, 2016. © 2016 Wiley Periodicals, Inc.

PRSS3 expression is associated with tumor progression and poor prognosis in epithelial ovarian cancer.

OBJECTIVE: PRSS3 is an atypical isoform of trypsin that has been associated with breast, lung, and pancreatic cancers. This study aimed to elucidate the role of PRSS3 in tumor tissues of patients with epithelial ovarian cancer (EOC) and to investigate the prognostic value of this marker. METHODS: PRSS3 expression was evaluated by immunohistochemistry and real-time PCR (RT-PCR) in ovarian cancers, benign ovarian tumors and the ovaries of age-matched normal patients. Correlations between clinicopathologic variables and PRSS3 expression in EOC tissues and the prognostic value of PRSS3 for progression-free survival (PFS) and overall survival (OS) were evaluated. RESULTS: PRSS3 expression was significantly elevated in EOC tissues compared to benign ovarian tumors and normal ovarian controls at both the mRNA and protein levels. There was a good correlation between the PRSS3 expression levels measured by the two different techniques. High PRSS3 expression in EOC tissues was significantly associated with advanced FIGO stage and lymph node metastasis. In a univariate survival analysis of the ovarian carcinoma cohort, positive expression of PRSS3 was significantly associated with shortened patient survival. Importantly, PRSS3 expression was a significant independent prognostic parameter in the multivariate analysis. CONCLUSIONS: These findings indicate that PRSS3 overexpression can be used as a predictor of clinical outcome in patients with ovarian cancer and may therefore represent a new prognostic marker.

Rational design of a novel propeptide for improving active production of Streptomyces griseus trypsin in Pichia pastoris.

Applying in silico simulations and in vitro experiments, the amino acid proline was proved to have a profound influence on Streptomyces griseus trypsinogen, and the hydrogen bond between H(57) and D(102) was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold.

Cyanobacterial protease inhibitors lead to maternal transfer of increased protease gene expression in Daphnia.

Protease inhibitors (PIs) have frequently been found in cyanobacterial blooms and have been shown to affect the major herbivore Daphnia by decreasing growth and inhibiting gut protease activity. However, it has been shown that a clone of Daphnia is able to respond to dietary PIs by increasing its protease gene expression. Such an inducible response might be maternally transferred to the next generation. Therefore, we tested a tolerant clone for maternal transfer of protease gene expression. When exposed to the trypsin inhibitor-producing cyanobacterium Microcystis aeruginosa PCC7806 Mut, Daphnia mothers and their untreated newborns showed an increase in trypsin gene expression compared to naïve mothers grown on control food and their offspring. The maternally transferred increase in gene expression was accompanied by a higher somatic growth rate of the offspring generation from exposed mothers compared to offspring from naïve mothers. This higher growth rate compensated for the lower dry mass of newborns from exposed mothers and led to the same fitness as observed in the offspring of naïve mothers. In nature, clones that can maternally transfer increased protease gene expression should have an advantage over clones that cannot. The selection for such more tolerant clones by naturally occurring PIs might lead to microevolution of natural Daphnia populations, and to local adaptation in the long term. This is the first study to show an adaptive maternal transfer of increased target gene expression in an ecological context.

[The effect of carbon tetrachloride poisoning on the activity of digestive proteases in rats and correction of the disorders with vegetable oils].

The results of the study of activity of digestive proteases (pepsin, trypsin, chymotrypsin) in homogenates of stomach, pancreas and duodenum in experimental animals have been presented. Rats were exposed to intoxication with carbon tetrachloride (subcutaneous administration of a 50% oil solution of CCl4 in the dose of 0.5 ml per 100 g body weight) for three days and then they were given analysed oils (black nut, walnut and flax oil) intragastrically by gavage at a dose of 0.2 ml per day within 23 days. Pepsin level in gastric mucosa homogenates and chymotrypsin activity in pancreatic homogenates were determined by method of N.P. Pyatnitskiy based on on the ability of enzymes to coagulate dairy-acetate mixture, respectively, at 25 degrees C and 35 degrees C. Trypsin activity in homogenates of pancreatic was determined by method of Erlanger - Shaternikova colorimetrically. It has been established that intoxication with CCl4 decreased the synthesis of proteolytic enzymes of the stomach (by 51%) and pancreas (by 70-78%). Injections of analysed vegetable oils to animals contributed to the normalization of proteolytic enzymes synthesis. The conclusion that there are prospects of using the analysed vegetable oils containing large quantity of polyunsaturated fatty acids (omega-3 and omega-6) for the correction of detected biochemical abnormalities has been done.

Tumour-associated trypsin inhibitor TATI is a prognostic marker in colorectal cancer.

BACKGROUND: The tumour-associated trypsin inhibitor TATI is expressed together with trypsin in many cancer forms, and an elevated serum level associates with poor prognosis. TATI can reduce tissue destruction by inhibiting trypsin and other proteinases, and in some cancer forms, its high tissue expression is associated with favourable prognosis. We analyzed the prognostic values of TATI, trypsinogen-1 and trypsinogen-2 immunoexpression from tissue array blocks constructed from surgical specimens of 592 colorectal cancer patients. RESULTS: TATI positivity correlated negatively with differentiation (p < 0.001) and positively with the histological type of adenocarcinoma (p < 0.001). Trypsinogen-1 and trypsinogen-2 positivity correlated with Dukes' stage (p = 0.045, p = 0.050); the percentage of trypsinogen-1- and trypsinogen-2-positive tumours was lower in metastasized (Dukes' stage C-D) than in local (Dukes' stage A-B) disease. In addition, trypsinogen-2 correlated inversely with differentiation (p = 0.012). In univariate analysis, the expression of TATI associated with more favourable cancer-specific survival (p = 0.010). In multivariate analysis, low TATI (p = 0.044), age (p < 0.001), Dukes' stage (p < 0.001), tumour differentiation (p = 0.020) and location in the rectum (p = 0.006) were independent prognostic factors for adverse outcome. Furthermore, TATI expression was an independent prognostic factor in a subgroup of trypsinogen-1- (p = 0.007) and trypsinogen-2-positive (p = 0.006) tumours. CONCLUSION: TATI tissue expression is an independent prognostic marker in colorectal cancer.

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris.

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6 U ml(-1) (batch cultivation with flask cultivation) or 14.4 U ml(-1) (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures.

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.

High Expression of PRSS3 Indicates Unfavorable Clinical Outcomes in Colon Adenocarcinoma.

INTRODUCTION: Serine proteases have been implicated as key drivers and facilitators of cancer malignancy. Protease, serine, 3 (PRSS3), which belongs to the serine proteases family, is reported to be abundantly expressed in a variety of types of tumor and contributes to the initiation and development of cancers. However, the clinical role of PRSS3 in colon adenocarcinoma (CAC) was not clarified yet. In the present study, we explored the potential effect of PRSS3 in CAC and whether it is related to the poor survival of CAC patients. MATERIALS AND METHODS: The mRNA and protein levels of PRSS3 were examined in CAC samples and connective noncancerous colon samples through quantitative real-time polymerase chain reaction assay and immunohistochemistry staining. Univariate and multivariate analyses were performed to estimate the prognostic role of PRSS3 in enrolled CAC patients. RESULTS: PRSS3 expression in CAC samples was significantly increased compared with connective noncancerous samples. Moreover, a higher level of PRSS3 was found to be correlated with the larger tumor size, advanced T stage, and positive lymph node metastasis. In addition, PRSS3 was also defined as an unfavorable prognosis factor for CAC patients. CONCLUSIONS: High expression of PRSS3 was significantly related to the unfavorable clinical features and poor prognosis in CAC patients. It suggested that PRSS3 might serve as a novel prognostic indicator and potential drug target for CAC treatment.

Cloning, heterologous expression and characterization of a novel streptomyces trypsin in Bacillus subtilis SCK6.

A novel streptomyces trypsin GM2938 was selected as the object of study. The active GM2938 contains 223 amino acid residues. Constructing recombinant plasmid and transforming Bacillus subtilis SCK6, the heterogenous expression of GM2938 was achieved. Through optimization of fermentation conditions, the expression level of GM2938 reached 1622.2 U/mL (esterase activity) and 33.8 U/mL (amidase activity). The recombinant trypsin was purified and measured: the specific activity of esterase was 5.6 × 103 U/mg, and the specific activity of amidase was 1.1 × 103 U/mg. Furthermore, the enzymatic properties of GM2938 were explore: the optimal reaction temperature and pH were 50 °C and 9.0, respectively; the recombinant enzyme show high stability at 25 °C and range of pH 5.0-9.0; Ca2+, K+, Mg2+, EDTA, DTT, DMSO, methanol, glycerin and ethanediol could promote the esterase and amidase activities at the investigated concentrations, while Fe2+, SDS, tritonx-100, acetone, chloroform and n-hexane inhibited the trypsin activities. Kinetic parameters of GM2938 were calculated: the Km of BAEE was 3.15 × 10-5 mol·L-1, Vmax value was 2.87 × 10-4 mol·L-1·min-1; the Km of BAPAN was 2.20 × 10-4 mol·L-1, the Vmax was 2.40 × 10-4 mol·L-1·min-1. These properties give trypsin GM2938 a potential application prospect.

Effect of sulfur mustard on melanogenesis in vitro.

The chemical warfare agent sulfur mustard (SM) affects all cells in the epidermis including melanocytes which are responsible for melanin synthesis. After exposure to SM, pigment abnormalities like hypo- and hyperpigmentation can occur. The underlying molecular pathomechanisms of SM exposure on human melanogenesis have not been elucidated so far. In our study, we investigated the effect of SM on human melanocytes and melanogenesis. Normal human epidermal melanocytes (NHEM) were used as in vitro model and they were exposed to different concentrations of SM (4.5 µM-100 µM). Melanin production was analyzed by absorption measurements at 405 nm. In addition, quantitative real-time PCR (qPCR) and Western blot experiments were performed to determine the expression of essential melanogenesis-related proteins including tyrosinase (TYR), tyrosinase-related protein (TRP) 1 and 2 and microphthalmia transcription factor (MITF). Our findings demonstrated that exposure to low SM concentrations increased melanin synthesis accompanied with an increase in protein expression. In contrast, high SM concentrations led to decreased melanin content and a downregulation in expression of all investigated melanogenesis-associated proteins. We concluded that low SM concentrations may cause hyperpigmentation while high SM concentrations decreased melanin content which may explain hypopigmented skin areas in SM exposed patients.

Redesigning trypsin: alteration of substrate specificity.

A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.

Isoleucine Regulates the Synthesis of Pancreatic Enzymes via the Activation of mRNA Expression and Phosphorylation in the Mammalian Target of Rapamycin Signalling Pathways in Pancreatic Tissues.

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.

Refolding and Activation from Bacterial Inclusion Bodies of Trypsin I from Sardine (Sardinops sagax caerulea).

BACKGROUND: Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. METHODS: TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. RESULTS: Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. CONCLUSION: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.