The preparation of 6x His-tagged granulocyte colony stimulating factor using an improved in vitro expression

An improved in vitro expression system called the rapid translation system (RTS) was used in this study for the in vitro biosynthesis of 6x His tagged granulocyte colony stimulating factor (6x His-tagged granulocyte colony-stimulating factor (GCSF)). This was done to overcome the problems associated with traditional cell based biotechnology. The study involved the preparation of template DNA for cell-free protein synthesis through gene amplification of open reading frame (ORF) of hGCSFb, cloning in pIVEX 2.4d vector and transformation of the produced construct in chemically competent Escherichia coli DH 5 α cells. A cell free protein synthesis system, RTS 100 E. coli HY kit, was tested for 6x His tagged G-CSF protein synthesis. Protein purification was done using Ni-NTA chromatography. Protein production was detected by two methods electrophoretically by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunologically by dot blotting immunodetection. The use of these methods yielded purified 6xHis-tagged GCSF with a concentration of about 250 μg/ml RTSreaction.Keywords: Granulocyte colony stimulating factor, in vitro expression system, RTS system

Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei. IV. Production in the fermentor

Optimization of the physical and physiological parameters of the fermentation process using the gene amplified variant of Amycolatopsis mediterranei (NCH) was carried out. Optimization of the physical parameters by controlling the pH at 6.5 for 3 days then at 7 thereafter and by adjustment of aeration at 1 vvm for 3 days then controlling the dissolved oxygen (DO) at 30% saturation increased the yield from 9.77 to 11.96 (22%) and 13.39 g/l (37%), respectively. Replacing 12% glucose in the fermentation medium (F2m1) with 5% glucose syrup (F2m3 medium) resulted in a drop of the yield from 9.77 to 7.5 g/l, while further addition of another 5% glucose syrup at day 4 increased the yield from 7.5 to 13.81 g/l (84%); with a further increase in the yield to 14.25 g/l (90%) upon controlling DO. Whereas, the combined addition of 0.1% yeast extract at day 2 to F2m3 medium along with the addition 5% glucose syrup at day 4 increased the yield from 7.5 to 15.35 g/l (105%); with a further increase in the yield to 16.3 g/l (117%) upon controlling DO. The fed-batch addition of both 3% soytone at day 3 and 5% of glucose syrup at day 4 to F2m3 medium increased the yield from 7.5 to 16.2 g/l (116%) and by extending the fermentation period to 10 days the yield reached 17.9 g/l (139%). Upon applying all optimum physical and physiological conditions in the fermentor the yield increased from 7.5 to 17.43 g/l in 8 days (132%) and by extending the fermentation period to 10 days the yield reached 19.4 g/l (159%). Further process optimization by examination and analysis of the kinetics of the process would most certainly further increase the yield and quantitatively define the process to a level that could be tested on a pilot scale. Key Words: Rifamycin B, fermentor, biotechnology, Amycolatopsis mediterranei, optimization, fed-batch and process development African Journal of Biotechnology Vol.3(9) 2004: 432-44

Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei. I. The role of colony morphology and nitrogen sources in productivity

A systematic approach to process optimization for production of rifamycin B was applied to a strain of Amycolatopsis mediterranei. Examination of the growth revealed 6 different morphologically distinct colonies on Bennett's agar medium. Rifamycin B production in shake flasks by the six different colony types ranged between 0.5 and 1.2 g/l. There was a clear correlation between the colony morphology and rifamycin B productivity. The highest yield of rifamycin B (1.03-1.2 g/l) was obtained by using the orange-red colored colonies, rosette shaped, devoid of hollow center and 2-3 mm in diameter. Variability in colony morphology, however, remained and the appropriate colonies had to be picked up for preparing the inoculum of each experiment. Addition of yeast extract to the fermentation medium at different times increased rifamycin B production. The highest antibiotic production was obtained upon the addition of 0.1% yeast extract after 2 days of incubation, where the yield increased from 1.15 to 1.95 g/l (70%). The use of 1.8% KNO3 in the fermentation medium, instead of 0.96% (NH4)2SO4, markedly increased rifamycin B production from 1.15 to 2.92 g/l (154%). It was also observed, upon microscopical examination, that KNO3 decreased branching and fragmentation of the mycelia in the fermentation medium. Keywords: Rifamycin B; fermentation; biotechnology; Amycolatopsis mediterranei; strain selection. African Journal of Biotechnology Vol.3(5) 2004: 266-27

Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei. II. The role of gene amplification and physiological factors in productivity in shake flasks

Amplification of gene expression of the most productive colony type of Amycolatopsis mediterranei strain N1 under stress of chloramphenicol, resulted in isolation of a variant NCH with productivity of 2.56 g/l compared to 1.15 g/l by the parent strain N1 (2.2 fold increase). This amplified variant has a further advantage of reduced variation in colony morphology with predominance of the most productive colony type. Using variant NCH, modification of the fermentation medium F1 by the addition of 0.1% yeast extract or the use of 1.8% KNO3 resulted in 3.8 and 5.8-fold increase in productivity, respectively, compared to strain N1. When the F1 medium was replaced by a new medium F2 containing soytone, instead of the particulate constituents (peanut meal and soybean meal) the yield by variant NCH reached 7.85 g/l (6.8-fold increase). Modification of the F2 medium by addition of glycerol or the replacement of glucose by glucose syrup decreased rifamycin B production. Changing the concentration of soytone increased the yield only slightly while replacing it with peptone or tryptone or the addition of 1 % corn steep liquor failed to increase the yield. On the other hand, the addition of 0.1 % yeast extract, or the replacement of 0.6% (NH4)2SO4 by 1.2% KNO3 or 0.4% NH4NO3, to F2 medium led to 8.2, 10.2 and 10.4-fold increase in productivity, respectively, compared to productivity of strain N1 in F1 medium. The change in the concentrations of either MgSO4 or CaCO3, the use of different types of antifoams and the use of higher concentrations of sodium diethyl barbiturate did not significantly influence the yield. These collective optimization attempts thus resulted in a 10.4-fold increase in productivity, from 1.15 to 11.99 g/l. Key words: Rifamycin B, fermentation, biotechnology, Amycolatopsis mediterranei, optimization, gene amplification, physiological factors. African Journal of Biotechnology Vol.3(5) 2004: 273-28

Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei. III. Production in fed-batch mode in shake flasks

Optimization of the fermentation process using the gene amplified variant of Amycolatopsis mediterranei, NCH, in the fermentation medium F2 was carried out by the application of fed-batch regime. The addition of 12% glucose alone at day 4 or simultaneously with 0.1% yeast extract at day 2 led to an increase in the yield of rifamycin B by 46% and 57%, respectively. The application of fed-batch regime together with replacing (NH4)2SO4 with the better yielding inorganic nitrogen sources NH4NO3 or KNO3 (F2m1 and F2m2 media, respectively) increased the production of rifamycin B. The use of F2m1 medium alone or with an additional 12% glucose added at day 4 increased the yield by 53% and 120%, respectively. However, further addition of 0.1% yeast extract led to an increase in the yield by only 63%. The addition of 3% soytone or 0.05% NH4NO3 to F2m1 at day 3 increased the yield by 72% and 61%, respectively, compared to productivity in F2 medium. The use of F2m2 medium increased the yield by 50%. The addition of 12% glucose at day 4 or of 0.1% yeast extract at day 2 to F2m2 medium led to an increase in the yield by 119 and 55%, respectively, compared to F2 medium. However, when both 12% glucose and 0.1% yeast extract were added at similar scheduled times only 64% increase in the yield occurred. By applying the three most effective optimization regimes determined using variant NCH with F2m2 medium on a standard rifamycin B producing strain, Nocardia mediterranei ATCC 21789, a similar pattern of increase in the antibiotic yield was observed. Thus, the use of F2m2 instead of F2 medium either alone or with an additional 12% glucose added at day 4 increased the yield by 36 and 75%, respectively, whereas the addition of 0.1% yeast extract to F2 medium increased the yield by 15%. In conclusion, the application of fed-batch technique with the optimum modifications of the medium constituents increased rifamycin B production by variant NCH to a maximum of 17.17 g/l compared to a yield of 5.3 g/l by the tested standard strain under the same conditions. The increase in rifamycin B production using the standard strain confirms the usefulness of the tested medium modifications in the improvement of rifamycin B production and its possible application in fermentations using other rifamycin B producer strains and also shows the superiority of variant NCH as a producer, when compared to the standard strain. Key words: Rifamycin B, fermentation, biotechnology, Amycolatopsis mediterranei, optimization, fed-batch and physiological factors. African Journal of Biotechnology Vol.3(8) 2004: 387-39

Impact of a package of diagnostic tools, clinical algorithm, and training and communication on outpatient acute fever case management in low- and middle-income countries: Protocol for a randomized controlled trial

Background: The management of acute febrile illnesses places a heavy burden on clinical services in many low- and middle-income countries (LMICs). Bacterial and viral aetiologies of acute fevers are often clinically indistinguishable and, in the absence of diagnostic tests, the ‘just-in-case’ use of antibiotics by many health workers has become common practice, which has an impact on drug-resistant infections. Our study aims to answer the following question: in patients with undifferentiated febrile illness presenting to outpatient clinics/peripheral health centres in LMICs, can we demonstrate an improvement in clinical outcomes and reduce unnecessary antibiotic prescription over current practice by using a combination of simple, accurate diagnostic tests, clinical algorithms, and training and communication (intervention package)? Methods: We designed a randomized, controlled clinical trial to evaluate the impact of our intervention package on clinical outcomes and antibiotic prescription rates in acute febrile illnesses. Available, point-of-care, pathogen-specific and non-pathogen specific (host markers), rapid diagnostic tests (RDTs) included in the intervention package were selected based on pre-defined criteria. Nine clinical study sites in six countries (Burkina Faso, Ghana, India, Myanmar, Nepal and Uganda), which represent heterogeneous outpatient care settings, were selected. We considered the expected seasonal variations in the incidence of acute febrile illnesses across all the sites by ensuring a recruitment period of 12 months. A master protocol was developed and adapted for country-specific ethical submissions. Diagnostic algorithms and choice of RDTs acknowledged current data on aetiologies of acute febrile illnesses in each country. We included a qualitative evaluation of drivers and/or deterrents of uptake of new diagnostics and antibiotic use for acute febrile illnesses. Sample size estimations were based on historical site data of antibiotic prescription practices for malarial and non-malarial acute fevers. Overall, 9 semi-independent studies will enrol a minimum of 21,876 patients and an aggregate data meta-analysis will be conducted on completion. Discussion: This study is expected to generate vital evidence needed to inform policy decisions on the role of rapid diagnostic tests in the clinical management of acute febrile illnesses, with a view to controlling the rise of antimicrobial resistance in LMICs. Trial Registration: Clinicaltrials.gov NCT04081051. Registered on 6 September 2019. Protocol version 1.4 dated 20 December 2019</p