Expression of anti-electronegative LDL single-chain fragment variable (scFv) in Pichia pastoris and its effect on foam cells formation

Author(s)

Soraya Megumi Kazuma

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2010-06-29

Examining board members

Dulcineia Saes Parra Abdalla; Irene da Silva Soares; Ana Marisa Chudzinski Tavassi

• Advisor: Dulcineia Saes Parra Abdalla

Abstract

The modification products of low-density lipoprotein (LDL), as the electronegative subfraction [LDL(-)], play an important role in the progression of atherosclerosis. The massive accumulation of modified LDL uptake by macrophages results in foam cells that release inflammatory mediators and contribute to atherogenesis. The scFv (singlechain fragment variable) is a recombinant antibody fragment that contains the complete site antigen-binding. Considering the role of LDL(-) in atherogenesis and the need for new therapeutic interventions that may inhibit the accumulation of lipids in macrophages, this study aimed the expression of anti-LDL(-) 2C7 scFv in Pichia pastoris and the evaluation of the effect of this recombinant antibody fragment on foam cells formation in cultured RAW 264.7 macrophages. The pPIgLE expression initial vector presented as a strategy for detection and purification the fusion with protein A. However, the high immunogenicity of the protein impairs the study of the fusion protein in cultured macrophages, leading to the replacement of the previous strategy of purification by chromatography with nickel resin by inserting hexahistidine tag at the C-terminus of the protein. The sequence analysis confirmed the presence of insertion and the complementary determining regions. The expression cassete with hexahistidine was inserted into the pPIgLE vector of P. pastoris and transformed in the SMD1168 strain (Invitrogen®). Preliminary tests of expression in small-scale allowed the analysis of seven different clones, showing a band corresponding to the molecular weight of 28KDa on SDS-PAGE, confirmed by Western Blot. The separation of 2C7 scFv by the nickel resin yield a pure protein, as it was shown by SDS-PAGE stained with silver. The affinity of 2C7 scFv was confirmed by Dot Blot. The assay of LDL(-) uptake showed that the 2C7 scFv was effective in reducing foam cells and this effect was determined by the decrease in gene expression of CD36, TLR-4 and COX-2. Based on these data, the 2C7 scFv demonstrates an important property for future therapeutic intervention for atherosclerosis (AU)

• FAPESP's process: 08/50487-5 - Expression of anti-electronegative LDL single-chain fragment variable (scFv) in Pichia pastoris and its effect on foam cells formation
• Grantee: Soraya Megumi Kazuma
• Support Opportunities: Scholarships in Brazil - Master