IMPORTANCE OF CODON PREFERENCE FOR PRODUCTION OF HUMAN RAP74 AND RECONSTITUTION OF THE RAP30 74 COMPLEX/

RAP30 and RAP74 are subunits of RAP30/74 (TFIIF, beta gamma), a genera l initiation and elongation factor for transcription by RNA polymerase II. Methods were previously published for production of human RAP30 a nd RAP74 in bacterial cells, using a bacteriophage T7 promoter express ion system. The vectors described for production of RAP74 were not ver y efficient and produced significant quantities of RAP74 amino termina l fragments. To improve these vectors, a segment of the human RAP74 cD NA was recoded using a preferred set of codons for translation in Esch erichia coil. Recoding dramatically improved protein production and su ppressed production of amino-terminal fragments. Improved vectors are reported that produce RAP74 with an LEHHHHHH carboxy-terminal extensio n (RAP74-H-6), for purification on a Ni2+-affinity column, and also wi th the native carboxy terminus (RAP74). Methods for purification of RA P74-H-6 and RAP74 are reported. Using these improved vectors, approxim ately 30 mg of soluble and active RAP74-H-6 or RAP74 can be produced a nd purified from 1 liter off. coil culture, representing a 10-fold imp rovement in protein production. Methods have also been developed for r econstitution of native RAP30/74 complex using recombinant proteins. T his complex has indistinguishable activity from human RAP30/74 for acc urate transcription in vitro. (C) 1994 Academic Press, Inc.