Properties of a cleaved two-chain form of recombinant human growth hormone.
Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.
Canova-Davis
E
E
Genentech, Inc., South San Francisco, California.
Baldonado
I P
IP
Moore
J A
JA
Rudman
C G
CG
Bennett
W F
WF
Hancock
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D013006:Growth Hormone; D004247:DNA