TABLE 1.

Recombinant production and one-step affinity purification of L. reuteri levansucrase from growth medium, using a B. megaterium expression system

Basic secretion vector	Final secretion vector	Encoded protein	Concn of secreted LevΔ773 (mg/liter culture)a	Concn of purified LevΔ773 (mg/liter culture)b
pMM1525	pMGBm4	LevΔ773	4.0
pHIS1525	pRBBm15	LevΔ773His	2.1	0.9d
pSTREP1525	pRBBm13	StrepLevΔ773	2.7	0.7c
pMM1525	pMMBm7	LevΔ773MycHis	1.6
pSTREPHIS1525	pRBBm16	StrepLevΔ773His	0.8

^aFor the induction of gene expression, 0.5% (wt/vol) xylose was added at an optical density at 578 nm of 0.4 to 500-ml cultures of B. megaterium MS941 carrying the indicated final, levansucrase-encoding plasmids. Cell-free growth medium was obtained by centrifugation. Protein amounts were calculated either via measured enzyme activity and specific activity or via densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from the growth medium.

^bA Bradford assay kit (Bio-Rad, Hercules, Calif.) was used for protein measurements.

^cAmmonium sulfate precipitation of proteins from cell-free growth medium was followed by StrepTactin affinity purification (IBA GmbH, Göttingen, Germany).

^dOne-step purification was achieved by batch incubation of Ni-nitrilotriacetic acid-Sepharose (Amersham-Pharmacia Biotech, Uppsala, Sweden) with cell-free growth medium prior to elution.