Secretion Expression E coli

Name	AA	DNA seq	Ref
DsbA	MKKI WLALAGLVL AFSASA	ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCG
OmpA	MKKTAIAIAVALAGFATVAQA
PelB	MKYLLP TAAAGLLLLA AQPAMA	ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCG
PhoA	MKQST IALALLPLL FTPVTKA	ATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCG
NSP4	MKKI TAAAGLLLLA AQPAMA	ATGAAAAAGATTACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCG	https://amb-express.springeropen.com/articles/10.1186/s13568-017-0394-1
OmpA + PelB	MKK LLPTAAAGLLLLAAQP		https://iopscience.iop.org/article/10.1088/1757-899X/301/1/012105/pdf
Is this going to work?	MKKI TAAAGLLLLA AQPAMG

PelB (pectate lyase B) from Erwinia carotovora MKYLLPTAAAGLLLLAAQPAMA

OmpA (outer-membrane protein A) MKKTAIAIAVALAGFATVAQA

StII (heat-stable enterotoxin 2) MKKNIAFLLASMFVFSIATNAYA

Endoxylanase from Bacillus sp. MFKFKKKFLVGLTAAFMSISMFSATASA

PhoA (alkaline phosphatase) MKQSTIALALLPLLFTPVTKA

OmpF (outer-membrane protein F) MMKRNILAVIVPALLVAGTANA

PhoE (outer-membrane pore protein E) MKKSTLALVVMGIVASASVQA

MalE (maltose-binding protein) MKIKTGARILALSALTTMMFSASALA

OmpC (outer-membrane protein C) MKVKVLSLLVPALLVAGAANA

Lpp (murein lipoprotein) MKATKLVLGAVILGSTLLAG

LamB (λ receptor protein) MMITLRKLPLAVAVAAGVMSAQAMA

OmpT (protease VII) MRAKLLGIVLTTPIAISSFA

LTB (heat-labile enterotoxin subunit B) MNKVKCYVLFTALLSSLYAHG

P00634 PPB_ECOLI Alkaline phosphatase Escherichia coli (strain K12) MKQSTIALALLPLLFTPVTKA
P02932 PHOE_ECOLI Outer membrane pore protein E Escherichia coli (strain K12) MKKSTLALVVMGIVASASVQA
P0A910 OMPA_ECOLI Outer membrane protein A Escherichia coli (strain K12) MKKTAIAIAVALAGFATVAQA
P02931 OMPF_ECOLI Outer membrane protein F Escherichia coli (strain K12) MMKRNILAVIVPALLVAGTANA
P09169 OMPT_ECOLI Protease 7 Escherichia coli (strain K12) MRAKLLGIVLTTPIAISSFA
P06996 OMPC_ECOLI Outer membrane protein C Escherichia coli (strain K12) MKVKVLSLLVPALLVAGAANA
P13811 ELBH_ECOLX Heat-labile enterotoxin B chain Escherichia coli MNKVKFYVLFTALLSSLCAHG
P02943 LAMB_ECOLI Maltoporin Escherichia coli (strain K12) MMITLRKLPLAVAVAAGVMSAQAMA
P0AEX9 MALE_ECOLI Maltose-binding periplasmic protein Escherichia coli (strain K12) MKIKTGARILALSALTTMMFSASALA
P0AEG4 DSBA_ECOLI Thiol:disulfide interchange protein dsbA Escherichia coli (strain K12) MKKIWLALAGLVLAFSASA
P0AEE5 DGAL_ECOLI D-galactose-binding periplasmic protein
Escherichia coli (strain K12) MNKKVLTLSAVMASMLFGAAAHA
P38683 TORT_ECOLI Periplasmic protein torT Escherichia coli (strain K12) MRVLLFLLLSLFMLPAFS
P0A855 TOLB_ECOLI Protein tolB Escherichia coli (strain K12) MKQALRVAFGFLILWASVLHA
P22542 HSTI_ECOLX Heat-stable enterotoxin II Escherichia coli (strain K12) MKKNIAFLLASMFVFSIATNAYA
P62593 BLAT_ECOLX Beta-lactamase TEM Escherichia coli MSIQHFRVALIPFFAAFCLPVFA
P00805 ASPG2_ECOLI l-asparaginase 2 Escherichia coli (strain K12) MEFFKKTALAALVMGFSGAALA
A2TJI4 CEXE_ECOLX Protein cexE Escherichia coli MKKYILGVILAMGSLSAIA
P05458 PTRA_ECOLI Protease 3 Escherichia coli (strain K12) MPRSTWFKALLLLVALWAPLSQA
P45523 FKBA_ECOLI FKBP-type peptidyl-prolyl cis–trans isomerase Escherichia coli MKSLFKVTLLATTMAVALHAPITFA
P69776 LPP_ECOLI Major outer membrane lipoprotein Escherichia coli (strain K12) MKATKLVLGAVILGSTLLAG
P31550 THIB_ECOLI Thiamine-binding periplasmic protein Escherichia coli (strain K12) MLKKCLPLLLLCTAPVFA
Q47537 TAUA_ECOLI Taurine-binding periplasmic protein Escherichia coli (strain K12) MAISSRNTLLAALAFIAFQAQA
P23857 PSPE_ECOLI Phage shock protein E Escherichia coli (strain K12) MFKKGLLALALVFSLPVFA
P07102 PPA_ECOLI Periplasmic appA protein Escherichia coli (strain K12) MKAILIPFLSLLIPLTPQSAFA
P34210 OMPP_ECOLI Outer membrane protease ompP Escherichia coli (strain K12) MQTKLLAIMLAAPVVFSSQEASA
P24093 DRAA_ECOL X Dr hemagglutinin structural subunit Escherichia coli (strain K12) MKKLAIMAAASMVFAVSSAHA
P0A915 OMPW_ECOLI Outer membrane protein W Escherichia coli (strain K12) MKKLTVAALAVTTLLSGSAFA
P0AFI5 PBP7_ECOLI D-alanyl-D-alanine endopeptidase Escherichia coli (strain K12) MPKFRVSLFSLALMLAVPFAPQAVA
P33590 NIKA_ECOLI Nickel-binding periplasmic protein Escherichia coli (strain K12) MLSTLRRTLFALLACASFIVHA
P0ADV1 LPTA_ECOLI Lipopolysaccharide export system protein lptA Escherichia coli (strain K12) MKFKTNKLSLNLVLASSLLAASIPAFA
P16397 SUBF_BACSU Bacillopeptidase F Bacillus subtilis MRKKTKNRLISSVLSTVVISSLLFPGAAGA
Q02113 CWBA_BACSU Amidase enhancer Bacillus subtilis MKSCKQLIVCSLAAILLLIPSVSFA
P34957 QOX2_BACSU Quinol oxidase subunit 2 Bacillus subtilis MVIFLFRALKPLLVLALLTVVFVLGG
O07921 CHIS_BACSU Chitosanase Bacillus subtilis MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA
P21130 SACB_BACAM Levansucrase Bacillus amyloliquefaciens MNIKKIVKQATVLTFTTALLAGGATQAFA
P39824 BLAC_BACSU Beta-lactamase Bacillus subtilis MKLKTKASIKFGICVGLLCLSITGFTPFFNSTHAEA
P07980 GUB_BACAM Beta-glucanase Bacillus amyloliquefaciens MKRVLLILVTGLFMSLCGITSSVSA
P31797 CDGT_BACST Cyclomaltodextrin glucanotransferase Bacillus stearothermophilus MRRWLSLVLSMSFVFSAIFIVSDTQKVTVEA
P06874 THER_BACST Thermolysin Bacillus stearothermophilus MNKRAMLGAIGLAFGLLAAPIGASA
P00808 BLAC_BACLI Beta-lactamase Bacillus licheniformis MKLWFSTLKLKKAAAVLLFSCVALAG
P06278 AMY_BACLI Alpha amylase Bacillus licheniformis MKQQKRLYARLLTLLFALIFLLPHSAAAA

Fermentor Culture Condition

https://amb-express.springeropen.com/articles/10.1186/s13568-017-0394-1 Small volume fed-batch cultures (3 mL working volume) were performed in a Micro 24 bioreactor system (Applikon Biotechnology, Forster City, CA, USA). Feed solutions and other supplements were sterilized by autoclaving or filtration through a 0.22-μm pore size filter, while the rich growth culture medium [20.3 g/L yeast extract (BioSpringer, Milwaukee, WI, USA), 10.1 g/L sodium sulfate anhydrous (JT baker, Center Valley, PA, USA) and 7 g/L K2HPO4 (JT baker, Center Valley, PA, USA)] was separately autoclaved and added later under aseptic conditions. The culture was initiated by inoculation of 2.8% culture volume into the rich growth medium containing 50 μg/mL of kanamycin (Sigma, St. Louis, MO, USA), 7.6 g/L of trace metal cocktail solution (55 g/L sodium citrate dehydrate (Sigma, St. Louis, MO, USA), 27 g/L FeCl3·6H2O (Sigma, St. Louis, MO, USA), 0.5 g/L CoCl2·6H2O (Sigma, St. Louis, MO, USA), 0.5 g/L Na2MoO4·2H2O (Sigma, St. Louis, MO), 0.95 g/L CuSO4·5H2O (Sigma, St. Louis, MO, USA), 1.6 g/L MnCl2·4H2O (Sigma, St. Louis, MO, USA), 1.3 g/L ZnCl2 (Sigma, St. Louis, MO, USA) and 2 g/L CaCl2 (Sigma, St. Louis, MO, USA) and 0.8% culture volume of the glycerol and Epsom salt solution [315 g/L of glycerol (Sigma, St. Louis, MO, USA) and 31.4 g/L of MgSO4 (Sigma, St. Louis, MO, USA)]. During the fed-batch cultivation, the impeller speed was initially set to 800 rpm and later controlled to keep the dissolved oxygen level (DO) at 60% saturation. In fed-batch mode, 55% (v/v) glycerol for the carbon source and 33% (w/v) yeast extract were used as feed solutions. Recombinant ATH35L gene expression was induced by addition of 0.5 mM isopropyl-β-D1-thiogalactopyranoside (IPTG) (Biovectra, Charlottetown, PE, USA) when the cell reached at an optical cell density (OD600) of 10. Cell culture was continued at 30 °C for an additional 14 h after induction. Cultured cells were collected at 14 h post-induction, and cell culture medium was collected from the harvest samples by centrifugation at 13,300g for 5 min for analysis and quantification of extracellular ATH35L. Periplasmic and cytoplasmic fractions were prepared from cell lysate from the harvest samples using PeriPreps™Periplasting kit (Epicentre, Madison, WI, USA).

Fermentor fed-batch culture processes Large volume fed-batch cultures were performed in a DasGip fermentor (SaniSure Inc, Moorepark, CA, USA) with 1 L working volume. Feed solutions, culture medium and other supplements were prepared as previously described for the small volume fed-batch culture processes. The culture was initiated by inoculation of 2.8% of culture volume into the prepared culture medium containing 50 μg/mL of kanamycin (Sigma, St. Louis, MO, USA), 7.6 g/L of trace metal cocktail solution, 15.8 g/L of glycerol (Sigma, St. Louis, MO, USA) and 1% (v/v) P2000 antifoam (Alfa Aesar, Reston, VA, USA) solution. Air space velocity was 1 vvm and the temperature was maintained at 30 °C. Ammonium hydroxide (23.5% v/v) (Sigma, St. Louis, MO, USA) and glacial acetic acid (50% v/v) solutions (Sigma, St. Louis, MO, USA) were used to maintain cultures at pH 7. During batch experiments, the impeller speed was initially set to 1200 rpm and later controlled to keep the DO at 60% saturation. In fed-batch mode, 55% (v/v) of glycerol for the carbon source and 33% (w/v) of yeast extract solutions were used as feed solutions. During feeding, the impeller speed was maintained constant at 1200 rpm, while the DO saturation was automatically kept at 60%. Recombinant ATH35L gene expression was induced by addition of 0.5 mM IPTG (Biovectra, Charlottetown, PE, USA) at an optical cell density of 80 (OD600). After induction, cell culture was continued in fed-batch mode at 30 °C for an additional 12 h. Cultured cells were collected at different time points post-induction to determine the profiles of secreted ATH35L protein, osmolality and the concentration of glycerol and acetate in culture medium. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427057/ https://www.frontiersin.org/articles/10.3389/fmicb.2019.01511/full

Strain  BL21 (DE3),  BL21 Star (DE3)

Medium

Seed medium (20 g/L yeast extract) Rich growth medium (20.3 g/L yeast extract, 10.1 g/L sodium sulfate anhydrous, 7 g/L K2HPO4

Shaker Incubator Medium

 Terrific Broth (TB) medium: glycerol 5g, tryptone 12g, yeast extract 24g,  K2HPO4 16.4g, and KH2PO4 2.3g. H2O--> 1L

Induction

grow OD600 to 0.8 , add 0.4 M IPTG, continue to grow 10 hours, add 0.4% Triton X-100, total induction time is 24 hours.