Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli.
Bovine somatotropin (bST) was secreted from Escherichia coli at moderate levels of 1-2 micrograms/ml/OD using expression vectors in which the bST gene was fused to the lamB secretion signal. To study the secretion properties of bST in E.coli further, two approaches for modifying the secretion signal were employed. In the first case, fusion proteins were constructed with six alternative bacterial secretion signals: three from E.coli proteins (HisJ, MalE and OmpA), two from bacteriophage proteins (M13 coat protein and PA-2 Lc) and one from the chitinase A protein of Serratia marcescens. The results, as monitored by Western blot analysis of both total cell protein and the periplasmic fraction, showed that these changes in the secretion signal did not significantly affect the secretion properties of bST. In the second approach, a library of random mutations was created in the lamB secretion signal and 200 independent clones were screened. The level of secreted bST was determined by growing individual clones in duplicate in microtiter wells, inducing protein expression and measuring the bST released by osmotic shock using a particle concentration fluorescent immunoassay. The secretion properties of several novel variants in the LamB signal peptide are presented.
Klein
B K
BK
Monsanto Corporate Research, Monsanto Company, St Louis, MO 63198.
Polazzi
J O
JO
Devine
C S
CS
Rangwala
S H
SH
Olins
P O
PO
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D004926:Escherichia coli / Q000502:physiology*; D005798:Genes, Bacterial; D013006:Growth Hormone / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone