Purification and characterization of recombinant mouse growth hormone binding protein produced in the baculovirus expression system

Protein Expression and Purification
G ThordarsonF Talamantes

Abstract

Sf21 insect cells were infected with recombinant baculovirus containing cDNA for the entire coding region of the mouse growth hormone binding protein (mGHBP). Recombinant (r) mGHBP was expressed at a yield of 17.3 mg/liter/3 days. The molecular size (Mr) of the rmGHBP was approximately 33,000 as estimated by SDS-PAGE. Amino-terminal sequence analysis of the recombinant protein yielded two sequences: one identical to amino acids l- 15 and another corresponding to amino acids 14-21 of the GHR/GHBP. Western blot analysis revealed that this is the same Mr as that of one of the two major Mr forms of serum mGHBP. Deglycosylation of serum mGHBP and recombinant mGHBP caused a shift in the molecular size of both proteins to that expected after removal of all N-linked carbohydrates. Binding characteristics of the recombinant mGHBP to mouse growth hormone were similar to those for serum GHBP. Scatchard analysis showed an equilibrium association constant (Ka) for rmGHBP of 3.8 x 10(8) +/- 0.6 x 10(8) M(-1) (mean +/- SEM, n = 3) and Ka of 9.2 x 10(8) +/- 2.0 x 10(8) M(-1) (mean +/- SEM, n = 3) for the serum mGHBP. In conclusion, this expression system should allow a production of relatively large quantities of mGHBP suitable for physiologic...Continue Reading

Citations

Dec 18, 2008·Journal of Peptide Science : an Official Publication of the European Peptide Society·Roberto M AguilarLuis S Haro
Mar 22, 2015·Matrix Biology : Journal of the International Society for Matrix Biology·Yoshifumi Itoh
Jul 29, 1998·Endocrinology·I G CamarilloF Talamantes
Dec 10, 1997·Journal of Chromatography. B, Biomedical Sciences and Applications·J Evangelista Dyr, J Suttnar

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