枯草芽孢杆菌表达系统

1Bacillus SubtilisExpression Vectors

Product Information and InstructionsNovember 20053generally regarded as safe); (ii) it has no significant bias in codon usage; (iii)it is capable of secreting functional extracellular proteins directly into the culturemedium (at present, about 60% of the commercially available enzymes areproduced by Bacillus species); (iv) a large body of information concerningtranscription, translation, protein folding and secretion mechanisms, geneticmanipulation and large-scale fermentation has been acquired.

But there are also two obstacles reducing the use of B. subtilis: (i) production ofa number of extracellular proteases which recognize and degrade heterologousproteins, and (ii) stable vector plasmids. The first obstacle has been largely solvedby the construction of protease-deficient strains. And the second has beencompletely overcome by introducing plasmids using the theta-mode of replicationsuch as those derived from the natural plasmids pAMβ1 and pBS72 (Jannière etal., 1990; Titok et al., 2003).

Quite recently, the construction and use of four different expression vectors basedon the E. coli - B. subtilis shuttle vector pMTLBS72 exhibiting full structural stabilitywas published (Nguyen et al., 2005).

The two new vectors pHT01and pHT43 allow high-level expression of recombinantproteins within the cytoplasm, where pHT43 directs the recombinant proteins intothe medium. Both vectors are based on the strong σA-dependent promoterpreceding the groE operon of B. subtilis which has been converted into anefficiently controllable (IPTG-inducible) promoter by addition of the lacoperator. Derivatives of pHT01 are available either with a 8xHis tag(pHT08), a Strep tag (pHT9) or a c-Myc tag (pHT10).pHT017955 bp

ORF-2AmpR

Pgrac

LacI

ORF-1

Cla IAfl II

Ahd I

ORF-3Sma IAat IIXba IBam HI

Sac I

CmRColE1Xho IApa IKpn IMun IBsp 119I

Bsp 119I5

pHT438057 bp

ORF-2AmpR

Pgrac

LacI

ORF-1Cla IAfl IIBsa I

ORF-3Sma IAat IIXba IBam HI

Sac I

CmRColE1Xho IApa IKpn IMun ISexAI

SamyQPgrac: Pgrac promoter (consisting of thegroE promoter; the lacO operator and thegsiB SD sequence)ColE1 ori: ColE1 originAmpR: ampicillin resistancelacI: lacI gene (lac repressor)CmR: chloramphenicol resistanceSamyQ: amyQ signal sequence

Complete DNA sequence is available onrequest.tgcgcggaagccatcaccatcaccatcaccatcacGGATCCTCTAGAgtcgacgtcCCCGGGLocation of the Strep tag in pHT09: Strep tagBamH I Xba I Sma I

Pgrac-lacO-RBS-atgaattggagccatccgcaatttgaaaaaGGATCCTCTAGAgtcgacgtcCCCGGG

Location of the c-Myc tag in pHT10: BamH I Xba I c-Myc tagPgrac-lacO-RBS-GGATCCTCTAGAgtcgacgtcgaacaaaaacttattagcgaagaagatctttaataacacgtc

3. ProtocolsDetailed protocols for E. coli and Bacillus molecular genetic handling(growth, transformation etc.) can be found in the relevant laboratory manualssuch as Sambrook and Russell (2001). For transformation of B. subtilis werecommend the protocol of Anagnostopoulos and Spizizen (1961), slightlymodified:

1.20 ml LS medium (Spizizen’s medium* supplemented with 0.5 %glucose, 5 µg/ml DL-tryptophane, 5 µg/ml uracil,0.01% casein hydrolysate, 0.1% yeast extract [Difco], 1 mM MgS04,2.5 mM MgCl2, 0.5 mM CaCl2) are inoculated with 1 ml of an 5 mlovernight culture grown in HS medium (Spizizen’s medium*supplemented with 0.5 % glucose, 50 µg/ml DL-tryptophane,50 µg/ml uracil, 0.02% casein hydrolysate, 0.1% yeast extract [Difco], 8µg/ml arginine, 0.4 µg/ml histidine, 1 mM MgSO4) at 37°C, shakingslowly at 30°C for 3 to 4 hours.

2.Incubate 1 ml of this HS culture (late log/early stationary phase; OD578)with 10 µl of 0.1 M EGTA at room temperature for 5 minutes and add 1to 2 µg plasmid DNA.

3.After shaking at 37°C for 2 hours for development of antibiotic resistance,the cells are plated on selective plates.

*Spizizen's medium: 2 g (NH4)2SO4, 14 g K2HPO4, 6 g KH2PO4, 1 g sodium citrate;add 100 ml distilled water, autoclave, then add 0.1 ml 1 M MgSO4.