Expression of cDNA fragment encoding sperm membrane peptide in E. coli.
A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the PR-PL promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. The E. coli cells transformed with pRSD-220 were propagated at 30 degrees C, then incubated at 42 degrees C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.
Shanghai Institute of Cell Biology, Academia Sinica, China.
Y
Y
Li
Y C
YC
Yan
W
W
Guo
F
F
Zhao
J
J
Lai
S S
SS
Koide
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013094:Spermatozoa / Q000378:metabolism*
D018076:DNA, Complementary; D008565:Membrane Proteins