High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1.
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.
Elleby
Björn
B
Department of Assay Development and Screening, Biovitrum AB, Stockholm SE-112 76, Sweden. bjorn.elleby@biovitrum.com
Svensson
Stefan
S
Wu
Xiaoqiu
X
Stefansson
Karin
K
Nilsson
Joakim
J
Hallén
Dan
D
Oppermann
Udo
U
Abrahmsén
Lars
L
D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D018834:Chaperonin 60 / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D009249:NADP / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins; D012995:Solubility
D018834:Chaperonin 60; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009249:NADP; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1