Influence of Ficoll on urea induced denaturation of fibrinogen

Ficoll is a neutral, highly branched polymer used as a molecular crowder in the study of proteins. Ficoll is also part of Ficoll-Paque used in biology laboratories to separate blood to its components (erythrocytes, leukocytes etc.,). Role of Ficoll in the urea induced denaturation of protein Fibrinogen (Fg) has been analyzed using fluorescence, circular dichroism, molecular docking and interfacial studies. Fluorescence studies show that Ficoll prevents quenching of Fg in the presence of urea. From the circular dichroism spectra, Fg shows conformational transition to random coil with urea of 6 M concentration. Ficoll helps to shift this denaturation concentration to 8 M and thus constraints by shielding Fg during the process. Molecular docking studies indicate that Ficoll interacts favorably with the protein than urea. The surface tension and shear viscosity analysis shows clearly that the protein is shielded by Ficoll

Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase.

The cofactor engineering strategy can relieve the metabolic stress induced by expression of recombinant protein in cellular metabolism related to cofactor and energy reactions. To study the effect of cofactor regeneration on recombinant protein expression, NADH oxidase (noxE) was engineered in P. pastoris expressing lipase B (GSCALB). Expression of noxE in P. pastoris (GSCALBNOX) increased NAD+ levels by 85% with a concomitant reduction in NADH/NAD+ ratio of 67% compared to GSCALB. The change in the redox level positively influenced the methanol uptake rate and made 34% augment in CALB activity. The decline in NADH level (44%) by noxE expression had lowered the adenylate energy charge (AEC) and ATP level in GSCALBNOX. In order to regenerate ATP in GSCALBNOX, adenylate kinase (ADK1) gene from S. cerevisiae S288c was co-expressed. Expression of ADK1 showed a remarkable increase in AEC and co-expression of both the genes synergistically improved CALB activity. This study shows the importance of maintenance of cellular redox homeostasis and adenylate energy charge during recombinant CALB expression in P. pastoris

Methanol utilisation pathway of <i>P</i>. <i>pastoris</i> highlighting the possible modes of regeneration of cofactors.

<p>AOX1/2 —Alcohol oxidase, FLD—formaldehyde dehydrogenase, CTA—catalase, FDH—formate dehydrogenase, DAS1/2 —dihydroxyacetone synthase, DAK—dihydroxyacetone kinase, FGH—s-formylglutathione hydrolase, FBA— 1,6 —bisphosphate aldolase, FBP—Fructose 1,6 bisphosphatase, noxE—NADH oxidase, ADK1 —adenylate kinase, DHA—dihydroxyacetone, GAP—glyceraldehyde 3 —phosphate, DHAP—dihydroxyacetone phosphate, GSH—glutathione, Xu— 5 P—xylulose 5—phosphate, F1,6BP—fructose 1,6, bisphosphate, F6-P—Fructose 6 –phosphate.</p

Comparison of relative gene expression levels of key metabolic genes and specific formaldehyde dehydrogenase enzyme activity in GSCALB and GSCALBNOX.

<p>Transcription of genes involved in methanol metabolism, glycolysis, TCA and glutamate catabolism were analysed by real- time PCR. The analysis was performed at 48, 72 and 96 h time intervals and the value represents the log₂ fold change calculated from three biological experiments. A: Relative expression of GSCALB compared to GS115 host, B: Relative expression of GSCALBNOX compared to GSCALB, C: Intracellular specific formaldehyde dehydrogenase enzyme activity.</p

Adenylate energy charge of GS115 host, GSCALB and GSCALBNOX.

<p>Adenylate energy charge of GS115 host, GSCALB and GSCALBNOX.</p

List of primers used in the study.

<p>List of primers used in the study.</p

Effect of noxE expression on CALB activity and methanol uptake rate.

<p>A: The CALB activity was analysed after methanol induction. B: Methanol uptake rate determined at every 12 h time intervals. GS115 host (▲), GSCALB (●) and GSCALBNOX (■). The error bars represents the deviation from the mean.</p

Effect of ADK1 gene expression on adenylate energy charge and CALB activity.

<p>Effect of ADK1 gene expression on adenylate energy charge and CALB activity.</p

Effect of noxE expression on intracellular nucleotide levels.

<p>The intracellular nucleotide levels were determined in GS115 host (▲), GSCALB (●), GSCALBNOX (■) at three—time points 48, 72 and 96 h. The level of NAD⁺ (A), NADH (B) and NADH/NAD⁺ (C) was determined by enzyme assay. The nucleotide analysis was carried out in triplicates and the error bar represents the deviation from the mean.</p