Advertisement

Expression of cloned cDNAs in mammalian cells from a cryptic promoter upstream to T7 in pGEM-4Z cloning vector

  • Shyam S. Chauhan
  • Puneet Seth
  • Rahul Katara
Article

Abstract

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.

Keywords

Transcription Mammalian expression vector Promoter cDNA Minigene pGEM-4Z 

Notes

Acknowledgments

This study was supported by grants from Council of Scientific and Industrial Research, and Department of Science and Technology, Government of India in favor of SSC. R.K. is the recipient of junior research fellowship from Indian Council of Medical Research, New Delhi, India.

References

  1. 1.
    Colosimo A, Goncz KK et al (2000) Transfer and expression of foreign genes in mammalian cells. Biotechniques 29:314–331PubMedGoogle Scholar
  2. 2.
    Craenenbroeck KV, Vanhoenacker et al (2000) Episomal vectors for gene expression in mammalian cells. Eur J Biochem 267:5665–5678. doi: 10.1046/j.1432-1327.2000.01645.x PubMedCrossRefGoogle Scholar
  3. 3.
    Doerfler W, Schubbert R, Heller H et al (1997) Integration of foreign DNA and its consequence in mammalian systems. Trends Biotechnol 15:297–301. doi: 10.1016/S0167-7799(97)01061-5 PubMedCrossRefGoogle Scholar
  4. 4.
    Yin W, Xiang P, Li Q (2005) Investigations of the effect of DNA size in transient transfection assay using dual luciferase system. Anal Biochem 346(2):289–294PubMedCrossRefGoogle Scholar
  5. 5.
    Olate J, Mattera R, Codina J et al (1988) Reticulocyte lysates synthesize an active alpha subunit of the stimulatory G protein Gs. J Biol Chem 263:10394–10400PubMedGoogle Scholar
  6. 6.
    Divya , Chhikara P, Chauhan SS et al (2002) Differential activity of cathepsin L in human placenta at two different stages of gestation. Placenta 23:59–64. doi: 10.1053/plac.2001.0748 PubMedCrossRefGoogle Scholar
  7. 7.
    Turk B, Turk D, Turk V (2000) Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 1477:98–111PubMedGoogle Scholar
  8. 8.
    Arora S, Chauhan SS (2002) Identification and characterization of a novel human cathepsin L splice variant. Gene 293:123–131. doi: 10.1016/S0378-1119(02)00700-X PubMedCrossRefGoogle Scholar
  9. 9.
    Chauhan SS, Popescu NC, Ray D et al (1993) Cloning, genomic organization, and chromosomal localization of human cathepsin L. J Biol Chem 268:1039–1045PubMedGoogle Scholar
  10. 10.
    Bakhshi R, Seth P, Chauhan SS et al (2001) Cloning and characterization of human cathepsin L promoter. Gene 275(1):93–101. doi: 10.1016/S0378-1119(01)00650-3 PubMedCrossRefGoogle Scholar
  11. 11.
    Seth P, Mahajan VS, Chauhan SS (2003) Transcription of human cathepsin L mRNA hCATL B from a novel alternate promoter in the first intron of the gene. Gene 321:83–91. doi: 10.1016/S0378-1119(03)00838-2 PubMedCrossRefGoogle Scholar
  12. 12.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory press, Cold Spring HarborGoogle Scholar
  13. 13.
    Smith SM, Gottesman MM (1989) Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli. J Biol Chem 264:20487–20495PubMedGoogle Scholar
  14. 14.
    Chauhan SS, Ray D, Kane SE et al (1998) Involvement of carboxy terminal amino acids in secretion of human lysosomal protease cathepsin L. Biochemistry 37:8584–8594PubMedCrossRefGoogle Scholar
  15. 15.
    Gal S, Gottesman MM (1988) Isolation and sequence of a cDNA for human pro-(cathepsin L). Biochem J 253(1):303–306PubMedGoogle Scholar
  16. 16.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467. doi: 10.1073/pnas.74.12.5463 PubMedCrossRefGoogle Scholar
  17. 17.
    Labow MA, Baim SB, Shenk T et al (1990) Conversion of a lac repressor into an allosterically regulated transcriptional activator for mammalian cells. Mol Cell Biol 10:3343–3356PubMedGoogle Scholar
  18. 18.
    Gottesman MM (1978) Transformation-dependent secretion of a low molecular weight protein by murine fibroblasts. Proc Natl Acad Sci USA 75(6):2767–2771. doi: 10.1073/pnas.75.6.2767 PubMedCrossRefGoogle Scholar
  19. 19.
    Rosfjord E, Lamb K, Rizzino A (1994) Cryptic promoter activity within the backbone of a plasmid commonly used to prepare promoter/reporter gene constructs. In Vitro Cell Dev Biol Anim 30:477–481. doi: 10.1007/BF02631317 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  1. 1.Department of BiochemistryAll India Institute of Medical SciencesNew DelhiIndia
  2. 2.Department of Molecular Genetics and MicrobiologyDuke University Medical CenterDurhamUSA

Personalised recommendations