Expression of porcine acid-labile subunit (pALS) of the 150-kilodalton ternary insulin-like growth factor complex and initial characterization of recombinant pALS protein.
Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S-transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.
Lee
Dong-Hee Lee
DH
Regional Animal Industry Research Center, Jinju National University, Jinju 660-758, Korea.
Chun
Choa
C
Kim
Sang-Hoon
SH
Lee
C Young
CY
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013552:Swine
D002352:Carrier Proteins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C075944:insulin-like growth factor binding protein, acid labile subunit