Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biotechnology and Applied Biochemistry (2001) 34, (25–32) (Printed in Great Britain)

PDF

Legacy HTML

Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column

Christin Andersson*, Marianne Hansson*, Ultan Power†, Per-Åke Nygren* and Stefan Ståhl*¹

*Department of Biotechnology, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden, and †Centre d'Immunologie Pierre Fabre, F-74 164 Saint-Julien en Genevois, France

Key words: affibody, affinity chromatography, mammalian cell expression, Semliki Forest virus, Staphylococcus aureus protein A.

Abbreviations used: BB, serum-albumin-binding region from streptococcal protein G; BHK-21 cells, baby hamster kidney cells; FCS, fetal calf serum; RSV, respiratory syncytial virus; G2Na, amino acids 130–230 of the G glycoprotein of RSV subgroup A; HSA, human serum albumin; SFV, Semliki Forest virus; SpA, Staphylococcus aureus protein A; SpG, streptococcal protein G; DEPC, diethyl pyrocarbonate.

¹To whom correspondence should be addressed (e-mail stefans@biochem.kth.se).

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

Received 8 January 2001/16 April 2001; accepted 25 April 2001

Portland Press Ltd ©2001