Article

Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells.

Oxford Protein Production Facility UK, Research Complex at Harwell, Rutherford Appleton Laboratory, Oxfordshire, UK.
Methods in molecular biology (Clifton, N.J.) 01/2012; 801:137-59. DOI:10.1007/978-1-61779-352-3_10
Source: PubMed

ABSTRACT In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

0 0
 · 
0 Bookmarks
 · 
71 Views
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

amplified DNA sequences
 
amplified sequences
 
bind
 
first framework sequence
 
heavy chains
 
heavy-chain genes
 
HEK 293T cells enables production
 
In-Fusion™ cloning
 
ligation-independent reaction
 
light-chain constant regions
 
mammalian cell expression vectors
 
mammalian cells
 
mixed 5' primers
 
protocols
 
recombinant Fabs
 
reverse transcription
 
Transient co-expression
 
transient expression
 
Variable region genes