Purification and characterization of recombinant mouse growth hormone binding protein produced in the baculovirus expression system.
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 physiological studies on the role of this protein.
Thordarson
G
G
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
Wu
K
K
Talamantes
F
F
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D013006:Growth Hormone / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011247:Pregnancy; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D018411:Spodoptera
D002352:Carrier Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013006:Growth Hormone; C051815:somatotropin-binding protein