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Table 1 Strains and plasmids used in this study

From: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

Strain or plasmid

Relevant characteristics

Source or reference

E. coli

 Stable

F’ proA + B + lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14-Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 ∆(mrr-hsdRMS-mcrBC)

New England Biolabs

 C43(DE3)

F– ompT gal dcm hsdSB(r −B m −B ) (DE3)

[55]

B. subtilis

 WB800N

nprE aprE epr bpr mpr::ble nprB::bsr∆vpr, wprA::hyg pB-cat5-neo-cat3 (NeoR)

[14]

 KO7

∆nprE,,∆aprE, ∆epr, ∆mpr, ∆nprB, ∆vpr, ∆bpr

Zeigler, D. R.; Bacillus Genetic Stock Center, Columbus, OH, USA

 KO7A

KO7, ∆wprA (HygR)

This work

Plasmids

 pBE-S

Vector used with the B. subtilis Secretory Protein Expression System, KmR, AmpR

TaKaRa/Clontech

 pET11M-senP2

Expression plasmid for SenP2 protease

H. Besir, EMBL Protein Expression and Purification Core Facility, Heidelberg, Germany

 pETM11-SUMO3GFP

T7 Expression vector for E. coli encoding His6-SUMO3-eGFP fusion protein, KanR

H. Besir, EMBL Protein Expression and Purification Core Facility, Heidelberg, Germany

 pET52B-senP2

SenP2 encoding region N-terminally fused to StrepII-tag

This work

 pHT01

IPTG-inducible expression vector for B. subtilis carrying Pgrac and CmR

[49]

 pJH-scFv

Phagemid cassette vector, IPTG-inducible expression of anti-GFP ScFv-pIII fusion protein, AmpR

This work

 pJHS01

pHT01 cured for MluI sites

This work

 pJHS02

pJHS01 encoding fusion protein consisting of AmyQ signal peptide, StrepII-tag, SUMO-tag and 6xHis-tag

This work

 pJHS03

pJHS02 with cloned phoA

This work

 pJHS04-07

pJHS03 with signal peptide encoding region changed (SPaprE (04), SPyncM (05), SPyopL,(06) and SPyoaW (07))

This work

 pJHS08

pJHS02 with cloned GFP-specific scFv gene

This work

 pJHS09

pJHS02 with SPamyQ changed for SPyoaW

This work

 pJHS10

pJHS09 with cloned GFP-specific scFv gene

This work

 pJHS11

pJHS09 with codon usage optimized SUMO-scFv fragment

This work

 pJHS12

pJHS09 with codon-usage optimized SUMO-cassette

This work

 pJHS13

pJHS02 with cloned barnase and barstar

This work

 pJHS14

pJHS12 with cloned barnase and barstar

This work

 pPA4

vector with cloned phoA

[50]

 pQEGFP

pQE30 (Qiagen) with cloned gfp

[53]

  1. SP signal peptide sequence