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 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-H-6), for purification on a Ni2+-affinity column, and also with the native carboxy terminus (RAP74). Methods for purification of RAP74-H-6 and RAP74 are reported. Using these improved vectors, approximately 30 mg of soluble and active RAP74-H-6 or RAP74 can be produced and purified from 1 liter off. 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. (C) 1994 Academic Press, Inc.