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-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.
Wang
B Q
BQ
Department of Biochemistry, Michigan State University, East Lansing 48824.
Lei
L
L
Burton
Z F
ZF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D005815:Genetic Code; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C033223:polyhistidine; D006639:Histidine; C056604:transcription factor TFIIF