Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-beta-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 microg/ml has increased the specific product yield of 66 mg optical density(-1) l(-1) (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-beta-1b obtained in this study is the highest that has been reported so far.

A purification method employing a process-control strategy was developed for improving the yield of rhG-CSF (recombinant human granulocyte colony-stimulating factor). A purity of >/=99% with an overall yield of 2.18 g/l was achieved in the present study. Analysis of the product during purification indicated that detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs (host cell proteins) without removing nucleic acid. Cysteine concentration was a key parameter in protein refolding. The bed height and HETP (height equivalent theoretical plates) value in the SEC (size-exclusion chromatography) column was evaluated and its impact on the resolution was studied. Formulation during SEC was found to be crucial for increasing the product yields with saving of time and process costs. The yield obtained in the present study is nearly four times higher than that reported in the literature. The product obtained was found to be acceptable for toxicological studies.

gyrB DNA fragments of seven Bacillus thuringiensis local collection family representatives were amplified by PCR and sequenced. Several differences in their corresponding sequences were evidenced. Both in silico and in vitro restriction maps of gyrB sequences and fragments respectively confirmed that EcoRI and Sau3AI could be used to differentiate between B. thuringiensis strains. However, the phylogeny analysis showed that only the gyrB PCR-Sau3AI allows a strains classification that correlates very well with that obtained on the basis of the sequences analysis. Thus, these finds show that gyrB PCR- Sau3AI digestion could be considered as an efficient, rapid, and easy method to make a distinction, not only between strains belonging to the Bacillus cereus group, but also between those belonging to B. thuringiensis.

The impact of different levels of agitation speed, carbondioxide and dissolved oxygen concentration on the key parameters and production of rhG-CSF in Escherichia coli BL21(DE3)PLysS were studied. Lower carbondioxide concentrations as well as higher agitation speeds and dissolved oxygen concentrations led to reduction in the acetate concentrations, and enhanced the cell growth, but inhibited plasmid stability and rhG-CSF expression. Similarly, higher carbondioxide concentrations and lower agitation speeds as well as dissolved oxygen concentrations led to enhanced acetate concentrations, but inhibited the cell growth and protein expression. To address the bottlenecks, a two-stage agitation control strategy (strategy-1) and two-stage dissolved oxygen control strategy (strategy-2) were employed to establish the physiological and metabolic conditions, so as to improve the expression of rhG-CSF. By adopting strategy-1 the yields were improved 1.4-fold over constant speed of 550 rpm, 1.1-fold over constant dissolved oxygen of 45%, respectively. Similarly, using strategy-2 the yields were improved 1.6-fold over constant speed of 550 rpm, 1.3-fold over constant dissolved oxygen of 45%, respectively.

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N'-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.