Expression of cloned cDNAs in mammalian cells from a cryptic promoter upstream to T7 in pGEM-4Z cloning vector.
In this study, we demonstrate that pGEM-4Z can be used as a mammalian expression vector. Western blotting and Immunocytochemical analyses revealed that transfection of pGEM-4Z-containing human cathepsin L cDNA under T-7 but not under SP-6 promoter into NIH 3T3 cells resulted in a high-level expression of cathepsin L. Expression of proteins using this vector in mammalian cells was further confirmed by using luciferase reporter gene. Furthermore, NIH 3T3 cells after stable or transient transfection with pGEM-4Z containing the first exon, first intron, and rest of the human cathepsin L cDNA downstream to its T-7 promoter synthesized and secreted large quantities of cathepsin L. RNase protection assays and 5' RACE established that the cloned cathepsin L cDNA is transcribed from a cryptic promoter present in the backbone of this vector upstream to T-7 sequence. This promoter was active in cell lines derived from four different mammalian species. In NIH 3T3 cells, this cryptic promoter could transcribe structural part of the genomic DNA into a primary transcript, which was efficiently spliced into mature mRNA and translated into protein. Thus this vector is equally useful for expressing proteins from genomic DNA. This hitherto unknown property of pGEM-4Z may be useful for expression of proteins in mammalian cells besides its use in synthesis of riboprobes, DNA sequencing, and in vitro transcription coupled translation assays.
Chauhan
Shyam S
SS
Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India. shyam_s_chauhan@yahoo.com
Seth
Puneet
P
Katara
Rahul
R
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection; D014709:Vero Cells
D018076:DNA, Complementary; D012333:RNA, Messenger; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse