Therapeutic protein expression platform of microbial system

A number of expression systems have been developed for the production of pharmaceutical products. Pichia pastoris and Escherichia coli expression system operate in our lab and express antibody fragment (scFv), cytokine, protein base adjuvant and vaccine and process enzyme. The expression platform are consisted of three part, first is strain generation , the second is fermentation process development in 250 ml fermentor and the last is process scale-up to 5 litter fermentor. Please click Additional Files below to see the full abstract

RNAMST: efficient and flexible approach for identifying RNA structural homologs

RNA molecules fold into characteristic secondary structures for their diverse functional activities such as post-translational regulation of gene expression. Searching homologs of a pre-defined RNA structural motif, which may be a known functional element or a putative RNA structural motif, can provide useful information for deciphering RNA regulatory mechanisms. Since searching for the RNA structural homologs among the numerous RNA sequences is extremely time-consuming, this work develops a data preprocessing strategy to enhance the search efficiency and presents RNAMST, which is an efficient and flexible web server for rapidly identifying homologs of a pre-defined RNA structural motif among numerous RNA sequences. Intuitive user interface are provided on the web server to facilitate the predictive analysis. By comparing the proposed web server to other tools developed previously, RNAMST performs remarkably more efficiently and provides more effective and flexible functions. RNAMST is now available on the web at

The microbial antibodies secretion expression platform with scale down fermentors

Therapeutic antibodies have become one of the most effective therapeutics for human diseases such as cancer, inflammation and viral infection. The production of antibody-based drugs using microbial expression systems is more cost effective with ease of gene manipulation compared to mammalian expression systems. In our team, antibody fragments (ex: BsAb, scFv and Fab) were produced from methylotrophic yeast Pichia pastoris secretion expression system with the AOX1 as driven promoter or E. coli secretion expression system. To achieve high production yield for both system, we investigated fermentation parameter such as base medium, induction medium, induction condition, feeding strategy and pH. For the 250 ml fermentor Pichia system, the nitrogen have been add into glycerol fed medium and/or methanol induction medium and also compared base-medium, buffered glycerol-complex medium (BMGY) and basal salt medium (BS). The highest scFv production was yielded from the basal salt medium as base medium, glycerol fed medium plus nitrogen and multiple carbon source methanol induction medium. This process can yielded over 500 mg/L scFv. After scale-up from 250 ml fermentor to 5L fermentor, the methanol fed-back control system also applied on the 5 L fermentor, can achieve 1.7 g/L scFv in 5 days. The E. coli expression process has passed through screening for high production yield clones in 2 ml deep-well then confirmed by using 250 ml flask scale. Feeding medium, DO, pH etc, parameters were investigated by parallel 250 ml-fermenter. The parameters from 250 ml fermentor were validated by using 5 L fermenter. Under this scale-up procedure, the antibody Fab was 100 folds production yield, production deep well stage at 1 mg/L, production from 250 ml fermentor stage is 50-100 mg/L and production 5 L fermentor stage is over 35-90 mg/L. Although different antibodies will result in different production yield, building a reliable platform to predict production yield from antibody cell clones under deep well and shake flask stage serves a good scale-down model for future scale-up prediction

Engineering of Escherichia coli protein expression process development

It almost 30% protein drugs are expression by Escherichia coli, because of rapid growth and high production yield. We have developed E.coli base system for recombinant protein expression, scFv, Fab and vaccine. In this study we introduce example about process development for nutrient components selection. Shaker flasks were used for different nitrogen and carbon components screening by DoE. Seven media formulations for E. coli fermentation were used in this study. By changing nitrogen and carbon source ratio, product titer of target protein could be optimized, at least 1.4 folds increased. The best result from shaker flask was used in 250 mL parallel fermenter and pH, dissolved oxygen, feeding/induction strategy were evaluated. The processes from seed culture to harvest only require 64 hours. The optimized time was reduced to 32 hours. The result showed that both target protein expression and cell density value were comparable, but the total process time was significantly reduced by half Please click Additional Files below to see the full abstract

Gram Level scFv expression platform of Pichia pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform. Please click Additional Files below to see the full abstract

Gram level scFv expression platform of Phichi pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform Please click Additional Files below to see the full abstract

α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding.

Invariant natural killer T (iNKT) cells, which are activated by T cell receptor (TCR)-dependent recognition of lipid-based antigens presented by the CD1d molecule, have been shown to participate in the pathogenesis of many diseases, including asthma and liver injury. Previous studies have shown the inhibition of iNKT cell activation using lipid antagonists can attenuate iNKT cell-induced disease pathogenesis. Hence, the development of iNKT cell-targeted glycolipids can facilitate the discovery of new therapeutics. In this study, we synthesized and evaluated α-lactosylceramide (α-LacCer), an α-galactosylceramide (α-GalCer) analog with lactose substitution for the galactose head and a shortened acyl chain in the ceramide tail, toward iNKT cell activation. We demonstrated that α-LacCer was a weak inducer for both mouse and human iNKT cell activation and cytokine production, and the iNKT induction by α-LacCer was CD1d-dependent. However, when co-administered with α-GalCer, α-LacCer inhibited α-GalCer-induced IL-4 and IFN-γ production from iNKT cells. Consequently, α-LacCer also ameliorated both α-GalCer and GSL-1-induced airway hyperreactivity and α-GalCer-induced neutrophilia when co-administered in vivo. Furthermore, we were able to inhibit the increases of ConA-induced AST, ALT and IFN-γ serum levels through α-LacCer pre-treatment, suggesting α-LacCer could protect against ConA-induced liver injury. Mechanistically, we discerned that α-LacCer suppressed α-GalCer-stimulated cytokine production through competing for CD1d binding. Since iNKT cells play a critical role in the development of AHR and liver injury, the inhibition of iNKT cell activation by α-LacCer present a possible new approach in treating iNKT cell-mediated diseases

The significance of seizures and other predictive factors during the acute illness for the long-term outcome after bacterial meningitis

SummaryBackgroundSeizures are important neurological complications of bacterial meningitis, but no information about its epidemiology and the outcomes of seizures after community-acquired bacterial meningitis (CABM) in an adult population have been reported.AimsTo determine the frequency, clinical relevance, subtypes of seizures during the acute phase of bacterial meningitis, and the long-term outcomes of seizure complicating adult CABM.MethodsIn this 12-year retrospective study, 117 adult patients were identified with culture-proven CABM. A comparison was made between the clinical data of the patients with and without seizures during hospitalization.ResultsThirty-one patients had seizures during CABM, accounting for 27% (31/117) of the episodes. The time interval between the onset of bacterial meningitis and the seizures was 1–21 days (mean, 4 days). Furthermore, 80% (25/31) of the episodes occurred within 24h of presentation. Ten patients who had seizures progressed to status epilepticus. At follow-up after completing treatment, 10 patients completely recovered and were seizure-free, 19 died of meningitis during the acute stage and the other two progressed to chronic epilepsy.ConclusionA log-rank test demonstrated that the long-term outcome of adult CABM with acute seizures produced worse outcomes than for those who had no seizures, though no difference was noted between focal and generalized seizures. None of our patients without seizures in the acute phase of bacterial meningitis developed late seizures during the follow-up periods. Poor outcome in this study may attribute to neurological complications such as seizure, hydrocephalus, infection itself, or a combination of complications

Multi-parametric neuroimaging evaluation of cerebrotendinous xanthomatosis and its correlation with neuropsychological presentations

<p>Abstract</p> <p>Background</p> <p>Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder. Recent studies show that brain damage in CTX patients extends beyond the abnormalities observed on conventional magnetic resonance imaging (MRI). We studied the MRI and ^{99 m}Tc-ethyl cysteinate dimer single photon emission computed tomography (SPECT) findings of CTX patients and made a correlation with the neuropsychological presentations.</p> <p>Methods</p> <p>Diffusion tensor imaging (DTI) and 3D T1-weighted images of five CTX patients were compared with 15 age-matched controls. Voxel-based morphometry (VBM) was use to delineate gray matter (GM) and white matter (WM) volume loss. Fractional anisotropy (FA), mean diffusivity (MD), and eigenvalues derived from DTI were used to detect WM changes and correlate with neuropsychological results. SPECT functional studies were used to correlate with GM changes.</p> <p>Results</p> <p>Cognitive results showed that aside from moderate mental retardation, the patient group performed worse in all cognitive domains. Despite the extensive GM atrophy pattern, the cerebellum, peri-Sylvian regions and parietal-occipital regions were correlated with SPECT results. WM atrophy located in the peri-dentate and left cerebral peduncle areas corresponded with changes in diffusion measures, while axial and radial diffusivity suggested both demyelinating and axonal changes. Changes in FA and MD were preceded by VBM in the corpus callosum and corona radiata. Cognitive results correlated with FA changes.</p> <p>Conclusion</p> <p>In CTX, GM atrophy affected the perfusion patterns. Changes in WM included atrophy, and axonal changes with demyelination. Disconnection of major fiber tracts among different cortical regions may contribute to cognitive impairment.</p