For the release of cellular or recombinant proteins into culture medium

products | DNA vector systems | BRP Plasmids & Competent BRP Cells

DNA Vector Systems

BRP Plasmids & Competent BRP Cells

Features

vectors expressing the Bacteriocin Release Protein (BRP)
protein excretion into E. coli culture medium without the need of a signal sequence
release of lethal proteins
preventing protein degradation by cytoplasmic proteinases
avoiding inclusion bodies
release of periplasmic proteins
large-scale biotechnological production of proteins in a continuous culture
simplified protein purification from culture medium
vectors can be co-transformed with ColE1 vectors
alternatively we offer competent BRP-transformed cells

Product Description:

The plasmids pJL3 and pSW1 express the Bacteriocin Release Protein (BRP), which initiates release of periplasmic and cytoplasmic E.coli proteins into the culture medium. Being compatible with most of the commonly used expression vector systems (e.g. ColE1 vectors like pAX, PheBo, pBR322 derivatives), BRP vectors can be co-transformed with the vector producing the recombinant protein of interest. Induction of the BR-Protein with IPTG (pJL3) or mitomycin C (pSW1) will cause an activation of phospholipase A in the outer E.coli membrane. This results in the formation of permeable zones in the cell membranes, through which proteins are released into the medium. A moderate induction prevents lysis of producer cells, also making the system suitable for large-scale protein production in a continuous culture. Since cloned proteins are no longer accumulated in the cytoplasm, problems associated with lethality of recombinant proteins, their preferential degradation or inclusion body formation are avoided.
For your convenience, we also provide competent cells already transformed with one of the vectors.

Cotransformation of recombinant plasmid and BRP vector (in one or two steps)

Induction of BRP, which then activates Phospholipase A

Excretion of cytosolic and periplasmic proteins into the cell culture medium

1. Cotransformation of recombinant plasmid and BRP vector (in one or two steps)

2. Induction of BRP, which then activates Phospholipase A

3. Excretion of cytosolic and periplasmic proteins into the cell culture medium

Examples:

Proteins, which have already been successfully released by activity of BRP from the E.coli periplasm are:

protein	size in kd
Bacillus penicillinase Aeromonas xylanase L Human IgG F_c region Human chimeric IgE/IgG F_c Human calcitonin Guar alpha -galactosidase Bacillus alpha -amylase Bacillus cellulase (N-4 and 1139) Human growth hormone Human tumor necrosis factor-alpha beta -lactamase FaeE	25 135 29 37 27 40 55 58/92 21 17 29 25

pJL3; p15A1 ori, origin of replication; Cm^R, chloramphenicol resistance; lpp^p, E. coli lipoprotein promoter, lac^po, lac promoter operator system; lac I, lac repressor.

pSW1; p15A1 ori, origin of replication; Tet^R, tetracycline resistance; Cm^R, part of chloramphenicol resistance gene, not functional; pClo, pCloDF13 promoter. BRP, Bacteriocin Release Protein.

ORDER INFORMATION, SHIPPING STORAGE:
order#	description	amount
BRPJL3	pJL3 vector DNA, lyophilized	5 µg
BRPSW1	pSW1 vector DNA,l yophilized	5 µg
shipped at RT; store at 4°C
COMJL3	Competent cells transformed with pJL3	5 x 200 µl
COMSW1	Competent cells transformed with pSW1	5 x 200 µl
shipped on dry ice; store at -70°C

Downloads: Handbook

For further Vector System products please download the Vector Systems brochure (PDF, 3,9 MB)

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