Codon optimization, expression, and characterization of recombinant lumbrokinase in goat milk.
Lumbrokinase is an important fibrinolytic enzyme derived from earthworm. Although its cDNA has been isolated and sequenced, there is still no report on expression of the lumbrokinase due to unknown reasons. To determine the elements affecting the expression of lumbrokinase, two copies of a lumbrokinase cDNA(w) obtained by RT-PCR and a synthesized lumbrokinase cDNA(m) with optimized codons were cloned into a mammary-gland-specific expression vector pIbCP. The pIbCP-LK-LK vector preparations were directly injected in the lactating goat mammary glands. Results showed that both LK-w and LK-m were successfully expressed in goat milk. The fibrinolytic activity of the LK-w in milk was 225,000 +/- 13,200 tPA units/L, while that of the LK-m was 550,000 +/- 21,600 tPA units/L, indicating that the codon optimization plays an important role in improving the lumbrokinase expression. The molecular weight of the recombinant lumbrokinase is 31.8 kDa. The main physiochemical features of the recombinant lumbrokinase, including temperature stability, pH resistance, and sensitivity to pepsin, were also clarified. This is the first report on expression and characterization of a genetically engineered lumbrokinase.
Laboratory of Genetic Engineering, Veterinary Institute, Academy of Military Medical Science, 5333 Xi'an Road, Changchun 130062, People's Republic of China. hurongliang@hotmail.com
Rongliang
R
Hu
Shoufeng
S
Zhang
Huiying
H
Liang
Ning
N
Li
Changchun
C
Tu
D000818:Animals; D003062:Codon; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005260:Female; D006041:Goats; D008892:Milk / Q000201:enzymology*; D009835:Oligochaeta / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D003062:Codon; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010450:Endopeptidases; C083421:lumbrokinase