Homologous Expression and Purification of Mutants of an Essential Protein by Reverse Epitope-Tagging


Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express non-complementing mutant enzymes in an E.coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5′ end of the chromosomal glnS-gene copy: this is referred to as ‘reverse epitope-tagging.’. The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Gold, L. 1990. Expression of heterologous proteins in Escherichia coli. Methods Enzymol. 185: 11–14.

    CAS  Article  Google Scholar 

  2. 2

    Williams, J.A., Langeland, J.A., Thalley, B.S., Skeath, J.B. and Carroll, S.B. 1995. Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies, p. 15–58. In: DNA Cloning 2: Expression Systems. Glover, D.M. and Hames, B.D. (Eds.). Oxford University Press Inc., New York.

    Google Scholar 

  3. 3

    Kast, P. and Hennecke, H. 1991. Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations. J. Mol. Biol. 222: 99–124.

    CAS  Article  Google Scholar 

  4. 4

    Auld, D.S. and Schimmel, P. 1995. Switching recognition of two tRNA synthetases with an amino acid swap in a designed peptide. Science 267: 1994–1996

    CAS  Article  Google Scholar 

  5. 5

    Munroe, S. and Pelham, H.R. 1987. A C-terminal signal peptide prevents secretion of luminal ER proteins. Cell 48: 899–907.

    Article  Google Scholar 

  6. 6

    Field, J., Nikawa, J.-I., Broek, D., MacDonald, B., Rodgers, L., Wilson, I.A., Lerner, R.A. and Wigler, M. 1988. Purification of a RAS-responsive adenylyl-cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8: 2159–2165.

    CAS  Article  Google Scholar 

  7. 7

    Kolodziej, P. and Young, R. 1989. RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus. Mol. Cell. Biol. 9: 5387–5394.

    CAS  Article  Google Scholar 

  8. 8

    Geli, V., Baty, D., Knibiehler, M., Lloubes, R., Pessegue, B., Shire, D. and Ladzunski, C. 1989. Synthesis and sequence-specific proteolysis of a hybrid protein (colicinA::growth hormone releasing factor) produced in Escherichia coli. Gene 80: 129–136.

    CAS  Article  Google Scholar 

  9. 9

    Sahin-Toth, M., Dunten, R.L. and Kaback, H.R. 1995. Design of a membrane protein for site specific proteolysis: properties of engineered factor Xa protease sites in the lactose permease of Escherichia coli. Biochemistry 34: 1107–1112.

    CAS  Article  Google Scholar 

  10. 10

    Uemura, H., Rogers, M.J., Swanson, R., Watson, L. and Söll, D. 1988. Site-directed mutagenesis to fine-tune enzyme specificity. Protein Engineering 2: 293–296.

    CAS  Article  Google Scholar 

  11. 11

    Wilson, I.A., Niman, H.L., Houghten, A.R., Cherenson, M.L., Connolly, M.L. and Lerner, R.A. 1984. The structure of an antigenic determinant in a protein. Cell 37: 767–778.

    CAS  Article  Google Scholar 

  12. 12

    Kulakauskas, S., Wikström, P.M. and Berg, D.E. 1991. Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome. J. Bacteriol. 173: 2633–2638.

    CAS  Article  Google Scholar 

  13. 13

    Bachmann, B.J. 1983. Derivations and genotypes of some mutant derivatives of Escherichia coli K12, p. 1190–1224. In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol 2. Neidhard, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M. and Umbarger, H. E. (Eds.). American Society of Microbiology, Washington, D.C.

    Google Scholar 

  14. 14

    Plumbridge, J. 1987. Organization of the Escherichia coli chromosome between genes glnS and glnU,V. Mol. Gen. Genet. 209: 618–620.

    CAS  Article  Google Scholar 

  15. 15

    Rogers, M.J., Oghi, T., Plumbridge, J. and Söll, D. 1988. Nucleotide sequence of the Escherichia coli nagE and nagB genes: The structural genes for the N-acetyl glucoseamine-6 phosphate deaminase. Gene 62: 197–207.

    CAS  Article  Google Scholar 

  16. 16

    Plumbridge, J. 1989. Sequence of the nagBACD operon in Escherichia coli K12 and pattern of transcription. Mol. Microbiol. 3: 505–515

    CAS  Article  Google Scholar 

  17. 17

    Wilson, K. 1992. Preparation of genomic DNA from bacteria, p. 2–10-2–12. In: Short Protocols in Molecular Biology (2nd Ed.). Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmann, J. G., Smith, J. A. and Struhl, K. (Eds.). John Wiley and Sons, New York.

    Google Scholar 

  18. 18

    Rould, M.A., Perona, J.J. and Steitz, T.A. 1991. Structural basis for anti-codon loop recognition by glutaminyl-tRNA synthetase. Nature 352: 213–218

    CAS  Article  Google Scholar 

  19. 19

    Conley, J., Sherman, J., Thomann, H.-U. and Söll, D. 1994. Domains of E. coli glutaminyl tRNA synthetase disordered in the crystal structure are essential for function or stability. Nucleosides and Nucleotides 13: 1581–1595

    CAS  Article  Google Scholar 

  20. 20

    Das, B.K., Bhattacharyya, T. and Roy, S. 1995. Characterization of a urea induced molten globule state of glutaminyl-tRNA synthetase from Escherichia coli. Biochemistry 34: 5242–5247

    CAS  Article  Google Scholar 

  21. 21

    Sherman, J.M. 1994. Specific recognition of tRNAs by aminoacyl-tRNA synthetases. Ph.D. Thesis, Yale University, New Haven, USA

  22. 22

    Carter, C.W., Jr. 1988. Cloning heterologous genes into Escherichia coli for enzyme production and crystal growth: problems of expression and micro-heterogeneity. Journal of Crystal Growth 90: 168–179

    CAS  Article  Google Scholar 

  23. 23

    Eriani, G. ., Delarue, M., Poch, O., Gangloff, J. and Moras, D. 1990. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347: 203–206

    CAS  Article  Google Scholar 

  24. 24

    Varadarajan, R., Szabo, A. and Boxer, S.G. 1985. Cloning, expression in Escherichia coli, and reconstitution of human myoglobin. Proc. Natl. Acad. Sci. USA 82: 5681–5684

    CAS  Article  Google Scholar 

  25. 25

    Carter, P., Abrahmsen, L. and Wells, J.A. 1991. Probing the mechanism and improving the rate of substrate-assisted catalysis by subtilisin BPN′. Biochemistry 30: 6142–6148

    CAS  Article  Google Scholar 

  26. 26

    Bieker, K.L. and Silhavy, T.J. 1989. PrlA is important for the translocation of exported proteins across the cytoplasmic membrane of Escherichia coli. Proc. Natl. Acad. Sci. USA 86: 968–972

    CAS  Article  Google Scholar 

  27. 27

    Romanos, M.A., Scorer, C.A. and Clare, J.J. 1995. Expression of cloned genes in yeast, p. 123–168. In: DNA Cloning 2: Expression Systems, Glover, D. M. and Hames, B. D. (Eds.). Oxford University Press Inc., New York

  28. 28

    Hopp, T., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, D.P., Urdal, D.L. and Conlon, P.J. 1988. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6: 1204–1210

    CAS  Article  Google Scholar 

  29. 29

    Hengen, P.H. 1995. Purification of His-Tag fusion proteins from Escherichia coli. Trends Biochem. Sci. 20: 285–286

    CAS  Article  Google Scholar 

  30. 30

    Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  31. 31

    Niman, H.L., Houghten, R.A., Walker, L.A., Reisfeld, R.A., Wilson, I.A., Hogle, J.M. and Lerner, R.A. 1983. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implication for the structural basis of immune recognition. Proc. Natl. Acad. Sci. USA 80: 4949–4953

    CAS  Article  Google Scholar 

  32. 32

    Harlow, E. and Lane, D. 1988 Antibodies, a Laboratory Manual. p. 521–523, Cold Spring Harbor Press, Cold Spring Harbor, NY

    Google Scholar 

  33. 33

    Winans, S.C., Elledge, S.J., Krueger, J.H. and Walker, G.C. 1985. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J. Bacteriol. 161: 1219–1221

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Yamao, F., Inokuchi, H., Cheung, A., Ozeki, H. and Söll, D. 1982. E. coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of the glnS-gene. J. Biol. Chem. 257: 11639–11643

    CAS  PubMed  Google Scholar 

  35. 35

    Barany, F. . 1985. New plasmids for selection of TAB linker mutations. Analects (Pharmacia Molecular Biology Division) 13: 5–6

  36. 36

    Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  37. 37

    Fersht A. 1985.Enzyme Structure and Mechanism (2nd. Ed.), Freeman and Company, New York, NY

    Google Scholar 

  38. 38

    Hoben, P. and Söll, D. 1985. Glutaminyl-tRNA synthetase of Escherichia coli. Methods Enzymol. 113: 55–59

    CAS  Article  Google Scholar 

  39. 39

    Kunkel, T.A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82: 488–492

    CAS  Article  Google Scholar 

  40. 40

    Schwob, E. and Söll, D. 1993. Selection of a ‘minimal’ glutaminyl-tRNA synthetase and the evolution of class I synthetases. EMBO J. 12: 5201–5208

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Dieter Söll.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Thomann, H., Ibba, M., Hong, K. et al. Homologous Expression and Purification of Mutants of an Essential Protein by Reverse Epitope-Tagging. Nat Biotechnol 14, 50–55 (1996). https://doi.org/10.1038/nbt0196-50

Download citation


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing