SpringerLink
Log in

Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter

  • Short Communication
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Escherichia coli is a widely used tool for the over-expression of human proteins for studying structure and function. The toxicity of human proteins for E. coli often hampers the expression. This study aims to find conditions for the expression of a membrane transporter known as the carnitine transporter CT2. The knowledge on this transporter is scarce, thus obtaining the recombinant protein is very important for further studies.

Methods and Results

The cDNAs coding for human CT2 (hCT2) was cloned in the pH6EX3 vector and different transformed E. coli strains were cultured in absence or in presence of glucose. hCT2 expression was obtained. The protein was purified and reconstituted into proteoliposomes in a functionally active state.

Conclusions

Using the appropriate IPTG concentration, together with the addition of glucose, hCT2 has been expressed in E. coli. The protein is active and shows capacity to transport carnitine in proteoliposomes. The results have a great interest in basic biochemistry of membrane transporters and applications to human health since hCT2 is involved in human pathology.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Bieber LL (1988) Carnitine. Annu Rev Biochem 57:261–283. https://doi.org/10.1146/annurev.bi.57.070188.001401

    Article  CAS  PubMed  Google Scholar 

  2. Huizing M, Iacobazzi V, Ijlst L, Savelkoul P, Ruitenbeek W, van den Heuvel L, Indiveri C, Smeitink J, Trijbels F, Wanders R, Palmieri F (1997) Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient. Am J Hum Genet 61(6):1239–1245. https://doi.org/10.1086/301628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pochini L, Scalise M, Galluccio M, Indiveri C (2013) OCTN cation transporters in health and disease: role as drug targets and assay development. J Biomol Screen 18(8):851–867. https://doi.org/10.1177/1087057113493006

    Article  CAS  PubMed  Google Scholar 

  4. Koepsell H, Lips K, Volk C (2007) Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res 24(7):1227–1251. https://doi.org/10.1007/s11095-007-9254-z

    Article  CAS  PubMed  Google Scholar 

  5. Pochini L, Galluccio M, Scalise M, Console L, Indiveri C (2019) OCTN: a small transporter subfamily with great relevance to human pathophysiology, drug discovery, and diagnostics. SLAS Discov 24(2):89–110. https://doi.org/10.1177/2472555218812821

    Article  CAS  PubMed  Google Scholar 

  6. Enomoto A, Wempe MF, Tsuchida H, Shin HJ, Cha SH, Anzai N, Goto A, Sakamoto A, Niwa T, Kanai Y, Anders MW, Endou H (2002) Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition. J Biol Chem 277(39):36262–36271. https://doi.org/10.1074/jbc.M203883200

    Article  CAS  PubMed  Google Scholar 

  7. Gong S, Lu X, Xu Y, Swiderski CF, Jordan CT, Moscow JA (2002) Identification of OCT6 as a novel organic cation transporter preferentially expressed in hematopoietic cells and leukemias. Exp Hematol 30(10):1162–1169. https://doi.org/10.1016/s0301-472x(02)00901-3

    Article  CAS  PubMed  Google Scholar 

  8. Sato N, Ito K, Onogawa T, Akahira J, Unno M, Abe T, Niikura H, Yaegashi N (2007) Expression of organic cation transporter SLC22A16 in human endometria. Int J Gynecol Pathol 26(1):53–60. https://doi.org/10.1097/01.pgp.0000225845.67245.b3

    Article  PubMed  Google Scholar 

  9. Carracedo A, Cantley LC, Pandolfi PP (2013) Cancer metabolism: fatty acid oxidation in the limelight. Nat Rev Cancer 13(4):227–232. https://doi.org/10.1038/nrc3483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wu Y, Hurren R, MacLean N, Gronda M, Jitkova Y, Sukhai MA, Minden MD, Schimmer AD (2015) Carnitine transporter CT2 (SLC22A16) is over-expressed in acute myeloid leukemia (AML) and target knockdown reduces growth and viability of AML cells. Apoptosis 20(8):1099–1108. https://doi.org/10.1007/s10495-015-1137-x

    Article  CAS  PubMed  Google Scholar 

  11. Zhao W, Wang Y, Yue X (2018) SLC22A16 upregulation is an independent unfavorable prognostic indicator in gastric cancer. Future Oncol 14(21):2139–2148. https://doi.org/10.2217/fon-2018-0207

    Article  CAS  PubMed  Google Scholar 

  12. Okabe M, Unno M, Harigae H, Kaku M, Okitsu Y, Sasaki T, Mizoi T, Shiiba K, Takanaga H, Terasaki T, Matsuno S, Sasaki I, Ito S, Abe T (2005) Characterization of the organic cation transporter SLC22A16: a doxorubicin importer. Biochem Biophys Res Commun 333(3):754–762. https://doi.org/10.1016/j.bbrc.2005.05.174

    Article  CAS  PubMed  Google Scholar 

  13. Novak AJ, Asmann YW, Maurer MJ, Wang C, Slager SL, Hodge LS, Manske M, Price-Troska T, Yang ZZ, Zimmermann MT, Nowakowski GS, Ansell SM, Witzig TE, McPhail E, Ketterling R, Feldman AL, Dogan A, Link BK, Habermann TM, Cerhan JR (2015) Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma. Blood Cancer J 5:e346. https://doi.org/10.1038/bcj.2015.69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bray J, Sludden J, Griffin MJ, Cole M, Verrill M, Jamieson D, Boddy AV (2010) Influence of pharmacogenetics on response and toxicity in breast cancer patients treated with doxorubicin and cyclophosphamide. Br J Cancer 102(6):1003–1009. https://doi.org/10.1038/sj.bjc.6605587

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lal S, Wong ZW, Jada SR, **ang X, Chen Shu X, Ang PC, Figg WD, Lee EJ, Chowbay B (2007) Novel SLC22A16 polymorphisms and influence on doxorubicin pharmacokinetics in Asian breast cancer patients. Pharmacogenomics 8(6):567–575. https://doi.org/10.2217/14622416.8.6.567

    Article  CAS  PubMed  Google Scholar 

  16. Faraji A, DehghanManshadi HR, Mobaraki M, Zare M, Houshmand M (2016) Association of ABCB1 and SLC22A16 gene polymorphisms with incidence of doxorubicin-induced febrile neutropenia: a survey of Iranian breast cancer patients. PLoS ONE 11(12):e0168519. https://doi.org/10.1371/journal.pone.0168519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kunii E, Oguri T, Kasai D, Ozasa H, Uemura T, Takakuwa O, Ohkubo H, Takemura M, Maeno K, Niimi A (2015) Organic cation transporter OCT6 mediates cisplatin uptake and resistance to cisplatin in lung cancer. Cancer Chemother Pharmacol 75(5):985–991. https://doi.org/10.1007/s00280-015-2723-x

    Article  CAS  PubMed  Google Scholar 

  18. Aouida M, Poulin R, Ramotar D (2010) The human carnitine transporter SLC22A16 mediates high affinity uptake of the anticancer polyamine analogue bleomycin-A5. J Biol Chem 285(9):6275–6284. https://doi.org/10.1074/jbc.M109.046151

    Article  CAS  PubMed  Google Scholar 

  19. Scalise M, Console L, Galluccio M, Pochini L, Indiveri C (2020) Chemical targeting of membrane transporters: insights into structure/function relationships. ACS Omega 5(5):2069–2080. https://doi.org/10.1021/acsomega.9b04078

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cosco J, Scalise M, Colas C, Galluccio M, Martini R, Rovella F, Mazza T, Ecker GF, Indiveri C (2020) ATP modulates SLC7A5 (LAT1) synergistically with cholesterol. Sci Rep 10(1):16738. https://doi.org/10.1038/s41598-020-73757-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bamber L, Harding M, Monne M, Slotboom DJ, Kunji ER (2007) The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes. Proc Natl Acad Sci USA 104(26):10830–10834. https://doi.org/10.1073/pnas.0703969104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Trinco G, Arkhipova V, Garaeva AA, Hutter CAJ, Seeger MA, Guskov A, Slotboom DJ (2021) Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by GltTk. Commun Biol 4(1):751. https://doi.org/10.1038/s42003-021-02267-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Setyawati I, Stanek WK, Majsnerowska M, Swier L, Pardon E, Steyaert J, Guskov A, Slotboom DJ (2020) In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters. Elife. https://doi.org/10.7554/eLife.64389

    Article  PubMed  PubMed Central  Google Scholar 

  24. Dulley JR, Grieve PA (1975) A simple technique for eliminating interference by detergents in the lowry method of protein determination. Anal Biochem 64(1):136–141. https://doi.org/10.1016/0003-2697(75)90415-7

    Article  CAS  PubMed  Google Scholar 

  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0

    Article  CAS  PubMed  Google Scholar 

  26. Galluccio M, Pantanella M, Giudice D, Brescia S, Indiveri C (2020) Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1). Mol Biol Rep. https://doi.org/10.1007/s11033-020-05717-8

    Article  PubMed  PubMed Central  Google Scholar 

  27. Holzhuter K, Geertsma ER (2022) Uniport, not proton-symport, in a non-mammalian SLC23 transporter. J Mol Biol 434(2):167393. https://doi.org/10.1016/j.jmb.2021.167393

    Article  CAS  PubMed  Google Scholar 

  28. Zhang L, Gui T, Console L, Scalise M, Indiveri C, Hausler S, Kullak-Ublick GA, Gai Z, Visentin M (2021) Cholesterol stimulates the cellular uptake of l-carnitine by the carnitine/organic cation transporter novel 2 (OCTN2). J Biol Chem 296:100204. https://doi.org/10.1074/jbc.RA120.015175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Console L, Scalise M, Mazza T, Pochini L, Galluccio M, Giangregorio N, Tonazzi A, Indiveri C (2020) Carnitine traffic in cells. Link with cancer. Front Cell Dev Biol 8:583850. https://doi.org/10.3389/fcell.2020.583850

    Article  PubMed  PubMed Central  Google Scholar 

  30. Scalise M, Pochini L, Giangregorio N, Tonazzi A, Indiveri C (2013) Proteoliposomes as tool for assaying membrane transporter functions and interactions with xenobiotics. Pharmaceutics 5(3):472–497. https://doi.org/10.3390/pharmaceutics5030472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Konstantinos Beis (Imperial College London) for supervising Tiziano Mazza during part of his PhD program.

Funding

This work was supported by “SI.F.I.PA.CRO.DE.–Sviluppo e industrializzazione farmaci innovativi per terapia molecolare personalizzata PA.CRO.DE.” Grant No. PON ARS01_00568 to CI granted by MIUR (Ministry of Education, University and Research) Italy.

Author information

Authors and Affiliations

  1. Department DiBEST Biologia, Ecologia, Scienze Della Terra Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci cubo 4C, 87036, Arcavacata di Rende, Italy

    Michele Galluccio, Tiziano Mazza, Mariafrancesca Scalise, Maria Chiara Sarubbi & Cesare Indiveri

  2. CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology IBIOM, via Amendola 122/O, 70126, Bari, Italy

    Cesare Indiveri

Authors
  1. Michele Galluccio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tiziano Mazza
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mariafrancesca Scalise
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Chiara Sarubbi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cesare Indiveri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cesare Indiveri.

Ethics declarations

Conflict of interest

All authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Galluccio, M., Mazza, T., Scalise, M. et al. Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter. Mol Biol Rep 49, 8185–8193 (2022). https://doi.org/10.1007/s11033-022-07491-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-022-07491-1

Keywords

Search

Navigation