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A method for in vitro synthesis of unglycosylated recombinant complement component C9

Taylor, Kathryn Mary, Luzio, J. P. and Campbell, Anthony Keith 1994. A method for in vitro synthesis of unglycosylated recombinant complement component C9. Journal of Immunological Methods 167 (1-2) , pp. 129-137. 10.1016/0022-1759(94)90082-5

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Abstract

A method for in vitrosynthesis of human complement component C9 has been established in order to generate unglycosylated normal and mutant proteins without the need to sub-clone. One or two step polymerase chain reaction (PCR) was used to add the T7 RNA polymerase promoter and introduce multiple mutations within the cDNA. The cDNA was then transcribed by T7 RNA polymerase and the mRNA translated in a rabbit reticulocyte lysate or wheat germ system. Successful synthesis was confirmed by: the correct size of PCR product DNA on agarose gel electrophoresis, incorporation of [α-32P]UTP into mRNA, and formation of [35S]methionine-labelled protein of the correct molecular mass for full length C9. The wheat germ extract generated up to 1.5 μg of recombinant C9. This unglycosylated C9 had at least 10% of the haemolytic activity of native C9. Unglycosylated C9 polymerised more readily than the native protein. This spontaneous polymerisation was increased by removal of the first 23 amino acids or mutating two cysteines at positions 33 and 36. This therefore provides a rapid method for screening the effect of multiple mutations on the biological activity and polymerisation of pore forming proteins.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Pharmacy
Subjects: Q Science > QD Chemistry
Q Science > QR Microbiology
R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords: Protein engineering; Complement C9; Polymerase chain reaction; Glycosylation
Publisher: Elsevier
ISSN: 0022-1759
Last Modified: 05 Jun 2017 02:58
URI: http://orca.cf.ac.uk/id/eprint/22487

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