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Protein Expr Purif. 1994 Oct;5(5):476-85.

Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex.

Author information

1
Department of Biochemistry, Michigan State University, East Lansing 48824.

Abstract

RAP30 and RAP74 are subunits of RAP30/74 (TFIIF, beta gamma), a general initiation and elongation factor for transcription by RNA polymerase II. Methods were previously published for production of human RAP30 and RAP74 in bacterial cells, using a bacteriophage T7 promoter expression system. The vectors described for production of RAP74 were not very efficient and produced significant quantities of RAP74 amino terminal fragments. To improve these vectors, a segment of the human RAP74 cDNA was recoded using a preferred set of codons for translation in Escherichia coli. Recoding dramatically improved protein production and suppressed production of amino-terminal fragments. Improved vectors are reported that produce RAP74 with an LEHHHHHH carboxy-terminal extension (RAP74-H6), for purification on a Ni(2+)-affinity column, and also with the native carboxy terminus (RAP74). Methods for purification of RAP74-H6 and RAP74 are reported. Using these improved vectors, approximately 30 mg of soluble and active RAP74-H6 or RAP74 can be produced and purified from 1 liter of E. coli culture, representing a 10-fold improvement in protein production. Methods have also been developed for reconstitution of native RAP30/74 complex using recombinant proteins. This complex has indistinguishable activity from human RAP30/74 for accurate transcription in vitro.

PMID:
7827505
DOI:
10.1006/prep.1994.1067
[Indexed for MEDLINE]

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