Challenges and Opportunities in the Process Development of Chimeric Vaccines.

Vaccines are integral to human life to protect them from life-threatening diseases. However, conventional vaccines often suffer limitations like inefficiency, safety concerns, unavailability for non-culturable microbes, and genetic variability among pathogens. Chimeric vaccines combine multiple antigen-encoding genes of similar or different microbial strains to protect against hyper-evolving drug-resistant pathogens.

Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts: A promising technology for SUMO fusion proteins.

The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli.

Effect of -glycosylation on secretion, stability, and biological activity of recombinant human interleukin-3 (hIL-3) in .

Human interleukin-3 (hIL-3) is a clinically important cytokine used to treat hematological malignancies, bone marrow transplantation, cytopenias, and immunological disorders. The cloning of hIL-3 gene was previously reported by our group, where its expression was optimized under methanol-inducible AOX1 promoter having N-terminal α mating factor signal sequence from . This study investigated the role of glycosylation pattern on its molecular stability, secretion efficiency, and biological activity using the mutagenesis approach.

Heterologous expression of novel SUMO proteases from Schizosaccharomyces pombe in E. coli: Catalytic domain identification and optimization of product yields.

Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus.

Development of a novel expression platform genomic integration of lipase gene for sustained release of methanol from methyloleate.

A novel Lip expression platform was developed by integrating lipase Lip2 from under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from was achieved utilizing methyloleate in LipAmyhost. Lipase hydrolyzed methyloleate into methanol that sustained P induction, and oleic acid, which was readily utilized as a carbon source.

Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris: A case study with proteolysis prone streptokinase.

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level.

L. rhizobacteria exhibit diversified cellulase and pectinase activities.

L. provides an enormous range of medicinal and nutritional benefits. The significant abilities of this plant to survive in Himalayan high altitudes enticed our study to investigate its rhizosphere.

Bioprocess optimization for the overproduction of catalytic domain of ubiquitin-like protease 1 (Ulp1) from S. cerevisiae in E. coli fed-batch culture.

The exploitation of SUMO (small ubiquitin-related modifier) fusion technology at a large scale for the production of therapeutic proteins with an authentic N-terminus is majorly limited due to the higher cost of ScUlp1 protease. Therefore, the cost-effective production of Saccharomyces cerevisiae Ulp1 protease catalytic domain (402-621 aa) was targeted via its cloning under strong T7 promoter with and without histidine tag. The optimization of cultivation conditions at shake flask resulted in ScUlp1 expression of 195 mg/L in TB medium with a specific product yield of 98 mg/g DCW.

Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris.

Streptokinase, a therapeutically important thrombolytic agent, is prone to C-terminal degradation and plasmin-mediated proteolytic processing. Since the protein was glycosylated during secretion from Pichia pastoris, therefore, the role of carbohydrate moieties on its stability was analyzed via in vivo blocking of N-glycosylation using tunicamycin where an increased degradation of streptokinase was observed. Further, the in vitro site-directed mutagenesis of the three putative N-glycosylation sites at asparagine residues 14, 265, and 377 to alanine revealed the essentiality of glycosylation of the 14th amino acid residue in its post-translational proteolytic stability without significantly affecting its biological activity.

High level extracellular production of recombinant γ-glutamyl transpeptidase from Bacillus licheniformis in Escherichia coli fed-batch culture.

Increasing demand of microbial γ-glutamyl transpeptidase (GGT) in food and pharmaceutical sectors raised the need for process development for high level production of the enzyme. In this respect, GGT from Bacillus licheniformis ER15 (SBLGGT) was cloned along with its native secretion signal and expressed in E. coli using different expression vectors.

Combined effect of gene dosage and process optimization strategies on high-level production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris fed-batch culture.

In this work, the combined effects of gene dosage and process optimization strategies were studied to achieve higher hIL-3 expression in Pichia system. The in-vitro multimerization method was used to generate various Pichia X-33 transformants having multi-copy expression cassettes. The quantitative polymerase chain reaction (qPCR) strategy was used to further confirm the genome integration of hIL-3 expression cassette.

A Malaria-Resistant Phenotype with Immunological Correlates in a Tanzanian Birth Cohort Exposed to Intense Malaria Transmission.

AbstractMalaria incidence is highly heterogeneous even in areas of high transmission, although no conclusive evidence exists that innate or naturally acquired resistance can prevent infection over an extended period of time. This longitudinal study examined immunoparasitological evidence for a malaria-resistant phenotype in which children do not develop malaria despite an extended period of exposure to parasites. Within a birth cohort followed from 2002 to 2006 in Muheza, Tanzania, an area of intense transmission, children ( = 687) provided blood smears biweekly during infancy and monthly thereafter.

High-level expression and efficient refolding of therapeutically important recombinant human Interleukin-3 (hIL-3) in E. coli.

Human interleukin-3 (hIL-3) is a pleiotropic cytokine that stimulates the differentiation and proliferation of multipotent hematopoietic cells thus making it a therapeutically important molecule. In this study, its poor expression yield was improved by addressing various upstream bottlenecks in E. coli heterologous system.

The evolution of recombinant thrombolytics: Current status and future directions.

Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage.

Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers.

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture.

Human interleukin-7 (hIL-7) is a therapeutically important cytokine involved in lymphocyte development and survival. In previous reports, a uniformly poor expression of hIL-7 has been shown in Escherichia coli host with the problem of inclusion body formation. In this study, the role of codon optimization and N-terminus blocking using various solubility enhancer fusion tags was explored to improve its soluble expression.

High level production of active streptokinase in Pichia pastoris fed-batch culture.

Streptokinase is a biological macromolecule involved in dissolution of fibrin blood clot and favourably used in various clinical applications. This protein is poorly expressed in soluble form due to its toxic effects on host physiology. The extracellular expression of recombinant streptokinase (SK) with and without 6xHis tag was obtained by cloning its gene under the α-mating factor signal sequence and alcohol inducible AOX1 promoter.

Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system.

The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.

Solid state bioconversion of wheat straw into digestible and nutritive ruminant feed by Ganoderma sp. rckk02.

Solid state fermentation (SSF) of wheat straw with Ganoderma sp. rckk02 was carried out for 15 days for improving its digestibility and nutrients. Fungal growth caused a significant (P<0.

Process development and cGMP manufacturing of a recombinant ricin vaccine: an effective and stable recombinant ricin A-chain vaccine-RVEc™.

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.

Optimization of human granulocyte macrophage-colony stimulating factor (hGM-CSF) expression using asparaginase and xylanase gene's signal sequences in Escherichia coli.

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E.

Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation.

Culture conditions for enhanced cellulase production from a newly isolated brown rot fungus, Fomitopsis sp. RCK2010 were optimized under solid state fermentation. An initial pH of 5.

Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion.

Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e.

White-rot fungal conversion of wheat straw to energy rich cattle feed.

In order to improve the digestibility and nutrient availability in rumen, wheat straw was subjected to solid state fermentation (SSF) with white-rot fungi (i.e. Pleurotus ostreatus and Trametes versicolor) and the fermented biomass (called myco-straw) was evaluated for biochemical, enzymatic and nutritional parameters.

Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics.

The biological delignification of lignocellulosic feedstocks, Prosopis juliflora and Lantana camara was carried out with Pycnoporus cinnabarinus, a white rot fungus, at different scales under solid-state fermentation (SSF) and the fungal treated substrates were evaluated for their acid and enzymatic saccharification. The fungal fermentation at 10.0 g substrate level optimally delignified the P.