Purification of human recombinant interleukin 1 receptor antagonist proteins upon Bacillus subtilis sporulation.
Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step.
Maurizi
G
G
Consorzio Biolaq, L'Aquila, Italy.
Di Cioccio
V
V
Macchia
G
G
Bossù
P
P
Bizzarri
C
C
Visconti
U
U
Boraschi
D
D
Tagliabue
A
A
Ruggiero
P
P
D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D002411:Cation Exchange Resins; D002455:Cell Division; D002458:Cell Fractionation; D002463:Cell Membrane Permeability / Q000502:physiology; D002474:Cell-Free System; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D008969:Molecular Sequence Data; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D013171:Spores, Bacterial
D002411:Cation Exchange Resins; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D012795:Sialoglycoproteins