Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris.
Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.
Grzelak
Krystyna
K
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Kłdkiewicz
Barbara
B
Kolomiets
Larissa I
LI
Debski
Janusz
J
Dadlez
Michał
M
Lalik
Anna
A
Ozyhar
Andrzej
A
Kochman
Marian
M
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000096:biosynthesis / Q000378:metabolism; D007915:Lepidoptera / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; D011994:Recombinant Proteins; C035606:juvenile hormone-binding protein, insect