A Leap Forward in Protein Production

Visit our Website Today!www.takara-bio.eu

Brevibacillus Expression

System

Protein Research Products

High Yields of Disulfide Bonded and Secreted Proteins

High Efficiency Secretion of Large Quantities of Proteins

Proteins are Produced in Biologically Active Forms

Good at Intracellular Production as well

www.takara-bio.eu phone:+33(0)139046880 email:info@takara-bio.eu

Brevibacillus choshinensis isagrampositivebacteriumwhichhasanexceptionalabilitytoexpressheterologousproteins.The New Brevibacillus Expression System, from Takara Bio, usesthebacteriumforhighefficiencysecretoryproduction.Thissystemproducesbotheukaryoticandheterologousproteins,inlargequantitiesandinactiveforms.AlsotheBrevibacillussystemisabletogeneratetheproteinwithaminoramountofextracellularproteasesotheproducedproteinisonlyslightlydegraded.SuccessfulresultsofproteinsexpressedcanbeseeninTable1.Thisincludesexpressionofenzymes,antigens,andcytokines.Eachproteinwasproducedataveryhighlevelofexpressionandconfirmedtohavenativebiologicalactivity.Inaddition,proteinsfromseveralorganismswhicharetaxonomicallyfarappart,suchaseubacteria,archaebacteria,eukaryotes,andviruses,weresuccessfullyproduced.Normallyeukaryoticproteinshaveintramolecularcysteinresiduesanddisulfidebondsmustbeformedatprecisepositionsmakingitdifficulttoobtainefficientproductionofproteinsusingintracellularbacterialexpressionsystems.TheBrevibacillussystemissuperiorwhenexpressingassemblingandexpressingsecretedeukaryoticproteins.Inaddition,B.choshinensisservesasanexcellenthostforintracellularproteinproduction.Furthermore,itproducesintracellularproteinsinasolubleforminthecytoplasmwithoutforminginclusionbodies.InseveralcasestheBrevibacillussystemworksbetterthanE.coliforexpression(Seepage3).Therefore,B. choshinensiscanbeusedasahostintheproductionofheterologousproteinsirrespectiveoforiginornature.

Brevibacillus Expression System

BioclipTMisaprocessthatusesEGFwhichcausessheeptoshedtheirfleece.DevelopedbyCSIROlivestockindustriesTheWoolmarkCompany.RecombinanthEGFisindustriallymanufacturedusingtheBrevibacillusexpressionsystem.

hEGF

Brevibacillus System Applications

Proteins Origins Production（ g/L ）Enzymes

α-amylase B. licheniformis 3.7Sphingomyelinase B. cereus 3Xylanase B. halodurans 0.2CGTase B. macerans 1.5Chitosanase B. circulans 1.4Hyper thermo-stable protease A. pernix 0.1Hyper thermo-stable nuclease P. horikoshii 0.7PDI human 1AntigensSurface antigen E. rhusiopathiae 0.9Surface antigen T. pallidum 0.8CytokinesEGF human 1.5IL-2 human 0.6NGF mouse 0.2IFN- γ chicken 0.5TNF-α bovine 0.4GM-CSF bovine 0.2GH flounder 0.2

Proteins Origins Production（ g/L ）Enzymesα-amylase B. licheniformis 3.7Sphingomyelinase B. cereus 3.0Chitosanase B. halodurans 0.2CGTase B. macerans 1.5Chitosanase B. circulans 1.4Hyperthermostable protease A. pernix 0.1Hyperthermostable nuclease P. horikoshii 0.7PDI human 1.0AntigensSurface antigen E. rhusiopathiae 0.9Surface antigen T. pallidum 0.8CytokinesEGF human 1.5IL-2 human 0.6NGF mouse 0.2IFN- γ chicken 0.5TNF-α bovine 0.4GM-CSF bovine 0.2GH flounder 0.2

Table 1: ExampleofproteinsexpressedusingtheBrevibacillusexpressionsystem

Construction of B. choshinensis Secretory Expression Plasmid

Activity of Expressed Protein

E.coli B. chosinensis

75_

50_

37_

kDa

sol solppt pptM C C C C1 1 1 1

3hr 16hrProtein Expression with B. choshinensis

75_

50_

37_

kDa

sol ppt

C C C C1 1 1 12 2 2 2

TMNm TMNm2SLNm 2SLNm

Brevibacillus versus E.coli Expression Systems

AcomparisonofintracellularproteinexpressionsystemsmeasuringeukaryoticDehydrogenase(DH)activitywasperformedusingE.coliwithapETvectorandtheBrevibacillusexpressionsystemusingthepNIvector.ThepNIvector,usedwiththeexpressionsystem,islackingasecretorysignalpeptidesotheexpressedproteinwillremaininthecytoplasm.TheexpressionintheBrevibacillussystemwasdeterminedusing2differentmedia.TheexpressionforE.colitestedtwodifferentinductiontimes.TheefficiencyofexpressionwasassessedthroughmeasurementofDHactivity(fig.1)aswellassolubilityoftheexpressedprotein(fig.2&3).

Fig. 1:DHactivitymeasurementofproteinexpressed,usingtheBrevibacillusexpressionsystem(in2differentmedia)orusingE.coli(after16hourinductionornoinduction).

Protein Expression with E.coli BL21 (DE3)

Fig. 2: ProteinexpressionusingE.coli BL21(DE3)after3and16hoursofinductioninsolublefractionaswellasprecipitate. M:Molecularweightmarker.C:pET-15b(control).1:pET-15b-DH.sol:solublefraction.ppt:precipitate.

Fig. 3:ProteinexpressionusingB. choshinensis in2differentmedia(TMNm&2SLNm)insolublefractionaswellasprecipitate.M:Molecularweightmarker.C:pNI(control).1-2:pNI-DH.sol:solublefraction.ppt:precipitate.

Recovery of Dehydrogenase activity is 10 to 20 times higher when expression is performed using the Brevibacillus expression system versus expression using E.coli BL21 (DE3). In conclusion, the Brevibacillus expression system produces a higher yield of active protein.

16h induction

- induction 2SLNm TMNm

SPP

PP Gene

Expressionvector

Expressionvector

＋Ligase

Stopcodon

SPcleavagesite

Gene

PCRFragment

：RestrictionSite

：Promoter

：Secretionsignalpeptide

：SDsequence

SP

SP

TaKaRa Bio recommends

High fidelity polymerase for PCR amplification of the target gene:PrimeSTAR HS DNA polymerase (R010A; R044A or R040A)

For efficient synthesis of long cDNA fragments: PrimeScript RTase (2680A or 6110A)

For protein purification:Talon Resin (635501 or 635606) or His60 (635659)

For insert cDNA cloning :DNA Ligation Kit <Mighty Mix> (6023) or DNA Ligation Kit Ver.2.1 (6022) and Takara’s restriction enzymes

Brev

ibac

illus

Exp

ress

ion

Syst

em

www.takara-bio.eu phone:+33(0)139046880 email:info@takara-bio.eu

Choosing a Brevibacillus Vector?

Vector Name Vector Type Expression Vector Lac Operator His-Tag Sec Signal Peptide Construct in Protease cleavage site

pNC-HisE (5,263 bp) Shuttle Vector Secretory Yes Yes Yes E.coli Enterokinase

pNC-HisT (5,260 bp) Shuttle Vector Secretory Yes Yes Yes E.coli Thrombin

pNC-HisF (5,260 bp) Shuttle Vector Secretory Yes Yes Yes E.coli Factor Xa

pNI-His DNA (5,079 bp) Shuttle Vector Intracellular Yes Yes NO E.coli Enterokinase

pNI DNA (5,055 bp) Shuttle Vector Intracellular Yes NO NO E.coli NO

pNY326 (3.4 Kb) Expression Secretory NO NO Yes Brevibacillus NO

pNCMO2 (5.2 kb) Shuttle Vector Secretory Yes NO Yes E.coli NO

pNCMO2-BLA Positive Control Secretory Has the gene encoding Bacillus licheniformis -amylase (55 kDa)

Vectors for the Brevibacillus System

Vectorssequencescanbefoundhere:http://www.takara-bio.com/products/download_e.htm

Product Name Catalog # Quantity BrevibacillusExpressionSystem HB100 1Kit Brevibacillus choshinensisElectro-cells HB115 100µLx10 pNC-HisEDNA HB123 10µg pNC-HisFDNA HB122 10µg pNC-HisTDNA HB121 10µg pNCMO2DNA HB112 10µg pNCMO2-BLADNA HB113 1µg pNIDNA HB131 10µg pNI-HisDNA HB132 10µg pNY326DNA HB111 10µg

Ordering Information

Product Name Catalog # Quantity Brevibacillus Expression system tik 001 BHKit Components

Expression vectors pNY326 DNA gµ 01111 BHpNCMO2 DNA gµ 01211 BHControl Vector pNCMO2-BLA DNA gµ 1311 BHCompetent cells (for electroporation)Brevibacillus choshinensis Electro-cells HB 115 100 µl x 10 tubes

Brevibacillus Expression System Kit

License Notices [L16] His-Tag Sequence This product is covered by the claims of U.S. Patent No. 5,284,933, 5,310,663 and their foreign counterpart patent claims. Protein Purification Technology of His-Tag used in some of pCold vectors and Brevibacillus expression vectors is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-La Roche Ltd., Basel, Switzerland and is provided only for the use in research. Information about licenses for commercial use is available from QIAGEN GmbH, Qiagen Strasse 1, D-40724 Hilden, Germany. [L39] Brevibacillus Expression System 1. B. choshinensis SP3, pNY326, pNCMO2, pNCMO2-BLA, pNC-HisT, pNC-HisF, pNC-HisE, pNI and pNI-His were developed and manufactured by Higeta Shoyu Co., Ltd. and sold by TAKARA BIO INC. 2. The use of this product is allowed for academic research only. You have to conclude a license agreement if you use this product or the products produced by using this product for commercial purpose. Contact us if you would like to ask any question about license etc. If any questions are received, either Higeta Shoyu Co., Ltd. or an agent for it will answer. TaKaRa Bio Inc. Tel +81-77-543-7247 Fax +81-77-543-9254 3. All rights of intellectual property related to this product are owned by Higeta Shoyu Co., Ltd. This product is covered by the following Japanese patents and corresponding international patent : Japanese patents, 2103774, 3696322, 3734593, 3753945, PCT/JP2004/016912 (international application based on PCT treaty), 3433807. 4. This product, the components or the derivatives of this product, or the products produced by using these items can not be transferred (distributed freely or sold) to third party. (When you purchase this product, you submit the license agreement to us.)Notice: All products are intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC.

All shuttle vectors between B. choshinensis andE.coli contain the P2 promoter, which is one of the five promoters that control transcription of the cell wall protein gene (HWP). This promoter does not work in E.coli but in B. choshinensis. The P2 promoter is quite robust and efficiently produces proteins.

The pNY326 vector* is maintained more stable than pNC or pNI vectors in the host cells due to much weaker promoter activity and to its smaller size (3.4 kb). Sometimes it works better than shuttle vectors, and is useful for a large scale production. The pNY326 vector must be constructed by the one step method using B. choshinensis. Brevibacillus choshinensis Electro-Cells(105 transformants/ug DNA) is used as the transforma-tion host.

*Can only be maintained in the B. choshinensis

P2 promoterLac operator

sec signal peptideHis-Tag

MCS

rep

Nmr

Ampr

ColE1 ori

pNC-HisT(5,260 bp)

P2 promoterLac operator

sec signal peptideHis-Tag

MCS

rep

Nmr

Ampr

ColE1 ori

pNC-HisF(5,260 bp)

P2 promoterLac operator

sec signal peptideHis-Tag

MCS

ori＋

rep

Nmr

Ampr

ColE1 ori

pNC-HisE(5,263 bp)

P2 promoterLac operator

pNI-His DNA(5,079 bp)

His-Tag P2 promoter Lac operator

pNI DNA(5,055 bp)

MCS MCSori -

ori＋

ori -

ori＋

ori -

ori＋

ori -

Nmr

Ampr

ori＋

ori -

Ampr

rep rep

Nmr

ColE1 ori ColE1 ori

rep

ori ＋

P5 promoterP2 promotersec signal peptide

X-terminator

pNY326（3.438 bp）

ColE1 ori

rep

ori ＋

pNCMO2(5.225 bp）

MCS MCSX-terminator

sec signal peptide

Nmr

NmrAmpr

ori - ori -

rep

ori ＋

P5 promoterP2 promotersec signal peptide

X-terminator

pNY326（3.438 bp）

ColE1 ori

rep

ori ＋

pNCMO2(5.225 bp）

MCS MCSX-terminator

sec signal peptide

Nmr

NmrAmpr

ori - ori -

TB633071EU