Secretion of eukaryotic growth hormones in Escherichia coli is influenced by the sequence of the mature proteins.
We report the construction of secretion plasmids expressing the fusion proteins, OmpA::pGH (pSpGH.01) and OmpA::hGH (phGH.01), and compare the secretion of mature porcine growth hormone (pGH) and human growth hormone (hGH) employing Escherichia coli. E. coli [phGH.01] secreted 10-15 micrograms hGH/ml/A600 cells into the periplasmic space, representing 30% of total periplasmic proteins. E. coli [pSpGH.01], however, secreted 30-fold less mature pGH. On the basis that both pSpGH.01 and phGH.01 are stably maintained in E. coli and in vitro transcription/translation data showed equivalent expression of OmpA::pGH and OmpA::hGH precursors, we attribute the higher secretion of hGH to the translocation-competent OmpA::hGH protein configuration. Two OmpA::GHF (growth hormone fusion) precursors, OmpA::GHF.02 and OmpA::GHF.03, both with hGH helix 3/helix 4 together instead of the pGH equivalent, secreted mature proteins as efficiently as OmpA::hGH. We propose that hGH helices 3 and 4 in these OmpA::GHF precursors play a major role in the folding of the precursor to a translocation-competent state, mimicking the translocation-competent nature of the OmpA::hGH precursor.
Cheah
K C
KC
Bresatec Ltd., Adelaide, Australia.
Harrison
S
S
King
R
R
Crocker
L
L
Wells
J R
JR
Robins
A
A
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D013552:Swine; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone