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
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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Wang BQ, Lei L, Burton ZF

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