Producing Recombinant mTEX101; a Murine Testis Specific Protein

Barzegar Yarmohammadi, Leila; Modarresi, Mohammad Hossein; Talebi, Saeed; Hadavi, Reza; Ostad Karampour, Mahyar; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

Producing Recombinant mTEX101; a Murine Testis Specific Protein

Authors

Leila Barzegar Yarmohammadi ¹

Mohammad Hossein Modarresi ²

Saeed Talebi ¹

Reza Hadavi ³

Mahyar Ostad Karampour ³

Ahmad Reza Mahmoudi ³

Mohammad Mehdi Akhondi ⁴

Hodjattallah Rabbani ⁵

Mahmood Jeddi-Tehrani ⁵

¹ Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran; Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Science, Tehran, Iran

² Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Science, Tehran, Iran; Pasteur Institute of Iran, Tehran, Iran

³ Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

⁴ Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

⁵ Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran; Immune and Gene Therapy Laboratory, Karolinska Cancer Center, Karolinska University Hospital, Stockholm, Sweden

Abstract

Introduction: Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous.Materials and Methods: RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a (+). The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 (DE3) E. coli strain.Results: A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies.Conclusion: We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies.

Keywords

Antibody production

gametogenesis

Germ cells

Glycoprotein

Recombinant protein

spermatogenesis

TES101 protein mouse

Testis

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