Addressing lipid impurities in Pichia pastoris cells producing virus like particles: genetic and bioprocess studies

Production of hepatitis B surface antigen (HBsAg) virus like particles (VLPs) occurs intracellularly in Pichia pastoris (P. pastoris). During extraction by cell disruption, host cell DNA and lipid impurities are released into the feed stream fouling the downstream process membranes and matrices. Effect of commercial triacylglycerol Candida rugosa (C. rugosa) lipase (CRL) on P. pastoris homogenate is investigated. P. pastoris homogenate was treated with CRL (0.0001 to 0.1 mg/mL) resulting in removal of triacylglycerol and increase in throughput by 60%, during constant flux filtration using 0.45μm polyethylene sulfone single layer disc membrane at 800 litres per square meter per hour. A strategy was employed where C. rugosa lipase 3 (CRL lip3) gene was localized in three different cellular locations (cytosol, endoplasmic reticulum, and cell surface) and encoded them under the control of PENO1 promoter alongside of the HBsAg gene under PAOX1. Developed strains were tested for expression of HBsAg along with optimisation of potentially toxic intracellular CRL lip3 expression. The CRL lip3 production did not influence HBsAg expression. A cell growth and gene induction regimes were identified with CRL lip3 localised to cell cytosol which enabled maximal HBsAg expression with independent control and CRL lip3 expression to peak only at the end of cultivation. Strains from the strategies which produced both, HBsAg as well as CRL lip3, were selected and further scaled-up to 0.25L bioreactor. The maximal sequential expression of HBsAg protein up to 20mg/L and CRL lip3 up to 150mg/L was confirmed. The formation of HBsAg VLP produced from co-expression culture was also confirmed using transmission electron microscopy. Downstream benefits of introducing lipases to VLP-containing P. pastoris homogenate and feasibility of cell engineering to produce CRL lip3 in a manner that does not otherwise compromise host cell performance was confirmed

A biological OR(XNOR) logic gate couples carbon source and transgene expression switching in a Komagatella phaffii (Pichia pastoris) strain co-producing process-enhancing lipase and a virus-like particle (VLP) vaccine

We constructed a three-input biological logic gate: S OR (G XNOR M), where S is sorbitol, G is glycerol, and M is methanol, to optimize co-expression of two transgenes in Komagataella phaffii using batch-mode carbon source switching (CSS). K. phaffii was engineered to harbor transgenes encoding a Candida rugosa triacylglycerol lipase, which can enhance downstream processing by removing host cell lipids from homogenates, and the hepatitis B virus surface antigen (HBsAg), a protein that self-assembles into a virus-like particle (VLP) vaccine. Using the native alcohol oxidase 1 (PAOX1) and enolase 1 (PENO1) promoters to direct VLP vaccine and lipase expression, respectively, successfully provided an OR(XNOR) gate function with double-repression as the output. This logic gate functionality enabled use of CSS to ensure that approximately 80% of total VLP yield was accumulated before cells were burdened with lipase expression in 250 mL DasGip bioreactor cultivation

Isolation, Purification and Partial Characterization of Crotalaria pallida Aiton Seed Proteins

Crotalaria pallida Aiton (Smooth rattlebox in English) is widely and wildly distributed in India. The seed of this plant is a valuable source of non-traditional proteins (about 22%) but attention was never been paid to explore the protein content. Chemical investigation has been conducted on Crotalaria pallida seeds (de-oiled) and proteins have been extracted in aqueous solution with different pHs or various concentrations of NaCl, KCl, Na2SO3 and CaCl2·2H2O at pH 7.0. The present study includes isolation, purification, and fractionation of seed protein along with its amino acid composition, molecular weight determination and surface topographies. Multiple polypeptide bands have been identified in the range of 16.5–61.6 kDa. The overall study confirms the seeds of this plant as an important source of unexplored protein