Methods for obtaining directionally truncated polypeptides

Details Methods for obtaining directionally truncated polypeptides Methods for obtaining directionally truncated polypeptides

2005-03-30

2010-06-01

Burkhart, Michael (Department: 1633)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using tissue cell culture to make a protein or polypeptide

C435S069100, C435S091410, C435S455000

Reexamination Certificate

active

07727744

ABSTRACT:
Methods, compositions and kits are disclosed for obtaining directionally truncated polypeptides by inserting a transposon. Preferably the transposon comprises a selectable marker and an ori, and optionally a promoter, a ribosome binding site and a translation start codon, into a target sequence in vitro or in vivo. Amplification products, varying in length depending on the transposon insertion site, are obtained using one primer that anneals to the target sequence and a second primer that anneals to the transposon. Amplification products are ligated to circular dsDNA, transformed into host cells, and individual colonies, each containing a directionally truncated clone of the target sequence, are obtained by plating on medium for which the selectable marker encodes resistance. Directionally truncated polypeptides encoded by the target sequence are obtained in vivo by inducing an RNAP in the host cells that uses the promoter or, in vitro by cell-free transcription and translation.

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