RESUMO
Fc optimization can significantly enhance therapeutic efficacy of monoclonal antibodies. However, existing Fc engineering approaches are sub-optimal with noted limitations, such as inappropriate glycosylation, polyclonal libraries, and utilizing fragment but not full-length IgG display. Applying cell cycle arrested recombinase-mediated cassette exchange, this study constructed high-quality monoclonal Fc libraries in CHO cells, displayed full-length IgG on cell surface, and preformed ratiometric fluorescence activated cell sorting (FACS) with the antigen and individual FcγRs. Identified Fc variants were quantitatively evaluated by flow cytometry, ELISA, kinetic and steady-state binding affinity measurements, and cytotoxicity assays. An error-prone Fc library focusing on the hinge-CH2 region was constructed in CHO cells with a functional diversity of 7.5 × 106. Panels of novel Fc variants with enhanced affinity and selectivity for FcγRs were isolated. Particularly, clone 2a-10 (G236E/K288R/K290W/K320M) showed increased binding strength towards FcγRIIa-131R and 131H allotypes with kinetic dissociation constants (KD-K) of 140 nM and 220 nM, respectively, while reduced binding strength towards FcγRIIb compared to WT Fc; clone 2b-1 (K222I/V302E/L328F/K334E) had KD-K of 180 nM towards FcγRIIb; clone 3a-2 (P247L/K248E/K334I) exhibited KD-K of 190 nM and 100 nM towards FcγRIIIa-176F and 176 V allotypes, respectively, and improved potency of 2.0 ng/ml in ADCC assays. Key mutation hotspots were identified, including P247 for FcγRIIIa, K290 for FcγRIIa, and K334 for FcγRIIb bindings. Discovery of Fc variants with enhanced affinity and selectivity towards individual FcγR and the identification of novel mutation hotspots provide valuable insights for further Fc optimization and serve as a foundation for advancing antibody therapeutics development.
RESUMO
Background: L-glutamic acid, the principal excitatory neurotransmitter in the brain and an important intermediate in metabolism acts as a precursor of γ-amino butyric acid (GABA). In the present study, culture condition for enhanced glutamic acid production by Lactobacillus plantarum MNZ was optimized and the influence of such conditions on GABA production was evaluated. Results: Results indicated that glutamic acid increased up to 3-fold (3.35) under the following condition: pH 4.5, temperature 37ºC, 12% (w/v) glucose and 0.7% (w/v) ammonium nitrate; whilst GABA production was enhanced up to 10-fold under the following condition: pH 4.5, temperature 37ºC, 6% (w/v) glucose and 0.7% (w/v) ammonium nitrate. Conclusions: This is the first report for dual biosynthesizing activities of a lactic acid bacterium for the production of glutamic acid and GABA. The results of this study can be further used for developing functional foods rich inglutamic acid and subsequently GABA as a bioactive compound.
Assuntos
Ácido Glutâmico/biossíntese , Lactobacillus plantarum/metabolismo , Ácido gama-Aminobutírico/biossíntese , Temperatura , Cromatografia Líquida de Alta Pressão , Ácido Glutâmico/análise , Ácido Butírico , Alimento Funcional , Fermentação , Compostos de Amônio , Ácido gama-Aminobutírico/análise , Glucose/análise , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Nitratos/metabolismoRESUMO
Aneurinibacillus thermoaerophilus strain AFNA as a novel isolated extracellular thermostable organic solvent tolerant lipase producing bacterium was employed in the present study. The lipase production of strain AFNA and its correlation with bacterial growth was studied via a modeling assessment by response surface methodology (RSM) and artificial neural network (ANN) techniques. The best achieved models were multilayer full feed forward incremental back propagation network and modified cubic response surface model (mRSM) using backward elimination. The highest lipase specific activity (13.1 Umg-1) and bacterial growth (OD600 = 3.0) were obtained at technically similar: growth temperature (53 and 53ºC), inoculum size (2.6 and 3.0 percent), agitation rate (118 and 115 rpm) and initial pH (7.0 and 7.2) but different medium volume (139 and 87 ml) and incubation period (48 and 38 hrs), respectively. In addition, the importance of effective parameters on the bacterial growth and lipase production was studied where pH and inoculum size were the most and the least effective factors, respectively. Significant correlation between lipase production and bacterial growth was observed when Bivariate correlation was employed to analyse the data. As a conclusion, lipase production was the result of a synergistic combination of effective parameters interactions and these parameters were in equilibrium.
Assuntos
Bacillaceae , Lipase/biossíntese , Redes Neurais de Computação , Modelos Teóricos , Propriedades de Superfície , TemperaturaRESUMO
Normal feed forward back-propagation artificial neural network (ANN) and cubic backward elimination response surface methodology (RSM) were used to build a predictive model of the combined effects and optimization of culture parameters for the lipase production of a newly isolated staphylococcus xylosus. The results demonstrated a high predictive accuracy of artificial neural network compared to response surface methodology. The optimum operating condition obtained from the ANN model was found to be at 30ºC incubation temperature, pH 7.5, 60 hrs incubation period, 1.8 percent inoculum size and 60 rpm agitation. The lipase production increased 3.5 fold for optimal medium. The produced enzyme was characterized biochemically and this is the first report about a mesophilic staphylococci bacterium with a high thermostable lipase which is able to retain 50 percent of its activity at 70ºC after 90 min and at 60ºC after 120 min. This lipase is also acidic and alkaline resistant which remains active after 24 hrs in a broad range of pH (4-11).