Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies

Protein Expression and Purification
Michal SharonJacob Anglister

Abstract

Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression syst...Continue Reading

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Citations

Jul 8, 2009·Protein Expression and Purification·Chenyun GuoDonghai Lin
Jul 24, 2003·Protein Science : a Publication of the Protein Society·Darrin A LindhoutBrian D Sykes
Jan 29, 2014·Journal of Peptide Science : an Official Publication of the European Peptide Society·Shuyan XiaoDaniel G S Capelluto
Aug 24, 2005·Biochemical and Biophysical Research Communications·Ganesan Senthil KumarSiddhartha P Sarma
Oct 20, 2006·Protein Expression and Purification·Hongjian LiJames Z Su
May 29, 2004·Protein Engineering, Design & Selection : PEDS·Takeshi TennoHidekazu Hiroaki

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