Preparation of Thermus thermophilus holo-chaperonin-immobilized microspheres with high ability to facilitate protein refolding.
Carboxylated poly(styrene/acrylamide) (P(St/AAm)-H) microspheres with different acrylamide contents were prepared by emulsifier-free emulsion polymerization. Thermus thermophilus holo-chaperonin (cpn) was covalently immobilized onto these microspheres with high yield. The T. thermophilus holo-cpn-immobilized microspheres were used for refolding of guanidine hydrochloride (Gdn-HCl)-denatured enzymes and showed sufficiently high ability to facilitate refolding of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PD) and pig heart lactate dehydrogenase (LDH) at 30 degrees C and Bacillus stearothermophilus LDH at 60 degrees C. The specific ability of T. thermophilus holo-cpn-immobilized microspheres increased with increasing immobilization amount and reached plateau at around 10-15 mg/m(2). When the immobilization amounts of T. thermophilus holo-cpn were approximately 10 mg/m(2), the microspheres retained sufficiently high ability to facilitate protein refolding during repeated use. Therefore, the P(St/AAm)-H microspheres on which approximately 10 mg/m(2) of T. thermophilus holo-cpn is immobilized are very effective for refolding of various (Gdn-HCl)-denatured enzymes over a wide temperature range.
Teshima
T
T
Division of Bioscience, The Graduate School of Science and Technology, Kobe University.
Kohda
J
J
Kondo
A
A
Taguchi
H
H
Yohda
M
M
Fukuda
H
H
D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D018833:Chaperonins / Q000737:chemistry*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D007934:Leuconostoc / Q000201:enzymology; D008863:Microspheres; D009206:Myocardium / Q000201:enzymology; D011137:Polystyrenes / Q000737:chemistry; D017510:Protein Folding; D020673:Protein Renaturation; D013552:Swine; D016963:Thermus thermophilus / Q000737:chemistry*; D000099166:Polyacrylamides
D000180:Acrylic Resins; D018833:Chaperonins; D005954:Glucosephosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase; D011137:Polystyrenes; D000099166:Polyacrylamides