Secretion of active bovine somatotropin in Escherichia coli.
We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.
Monsanto Corporate Research, Monsanto Co., St. Louis, MO 63198.
B K
BK
Klein
S R
SR
Hill
C S
CS
Devine
E
E
Rowold
C E
CE
Smith
S
S
Galosy
P O
PO
Olins
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D018272:Porins; D011401:Promoter Regions, Genetic; D011869:Radioligand Assay; D011938:Rec A Recombinases / Q000235:genetics; D011991:Receptors, Virus / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping
D001425:Bacterial Outer Membrane Proteins; D018272:Porins; D011991:Receptors, Virus; D011994:Recombinant Proteins; C016532:maltoporins; D013006:Growth Hormone; D011938:Rec A Recombinases