Host strain

E. coli strain for producing recombinant proteins incorporating synthetic amino acids

  • Dr. Kensaku Sakamoto and his colleagues at RIKEN Center for Biosystems Dynamics Research (RIKEN BDR) developed host E. coli strains that can produce recombinant proteins incorporating synthetic amino acids by reassignment of the UAG codon.
  • The RFzero-iy strains are designated to incorporate 3-iodotyrosine into produced proteins. For the incorporation, the strain is transformed with the expression plasmid in which a codon for the target tyrosine is replaced with UAG codon and cultured in 3-iodotyrosine containing medium (Mukai, T. et al., 2011). Two strains based on the BW25113 and BL-21(DE3) are available.
  • The B-95.deltaA strains can be assigned their UAG codon to synthetic amino acids by the introduction of plasmids carrying specific pair of UAG-reading tRNA and an aminoacyl-tRNA synthetase (aaRS) variant*. Increased productivity of sulfonated hirudin which is expected to improve the inhibition of blood coagulation was reported (Mukai, T. et al., 2015). The original and the growth improved derivative strains are available.
  • Web site
    Incorporation of synthetic amino acids into proteins at specific sites
  • Genetic-code evolution for protein synthesis with non-natural amino acids.
    Mukai T, Yanagisawa T, Ohtake K, Wakamori M, Adachi J, Hino N, Sato A, Kobayashi T, Hayashi A, Shirouzu M, Umehara T, Yokoyama S, Sakamoto K.
    Biochem. Biophys. Res. Commun. 411 (4): 757-761, 2011. [PMID 21782790]
    Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.
    Mukai T, Hoshi H, Ohtake K, Takahashi M, Yamaguchi A, Hayashi A, Yokoyama S, Sakamoto K.
    Sci. Rep. 5: 9699, 2015. [PMID 25982672]

    Catalog# Resource name Description
    RDB13711 B95. delta A Escherichia coli BL-21(DE3)-based host strain with no specific assignment of the UAG codon.
    RDB13712 B95. delta A delta fabR Escherichia coli BL21(DE3)-based host strain with no specific assignment of the UAG codon.
    RDB14427 BW25113-based RFzero-iy Escherichia coli host strain to produce non-natural amino acids containing proteins.
    RDB14428 BL21(DE3)-based RFzero-iy Escherichia coli host strain to produce non-natural amino acids containing proteins.
  • Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates.
    Yamaguchi A, Matsuda T, Ohtake K, Yanagisawa T, Yokoyama S, Fujiwara Y, Watanabe T, Hohsaka T, Sakamoto K.
    Bioconjug. Chem. 27 (1): 198-206, 2015. [PMID 26625213]

    Catalog# Resource name Description
    RDB20115 pCDF-Az Expression vector of the azidophenylalanine-specific variant of Methanocaldococcus jannaschii TyrRS and its cognate amber suppressor tRNA.
    RDB20116 pCDF-Mm2 Expression vector of the variant of Methanosarcina mazei PylRS specific for pyrrolysine derivatives (Boc-Lys, Az-ZLys, AzAmZLys) and its cognate Pyl tRNA.

E. coli auxotroph host strain used for selective isotope labeling

  • All ML strains werre engineered by Dr. Gennis research group at University of Illinois at Urbana-Champaign, U.S.A., and YM, MS, EH, and RF strains were engineered by Dr. Iwasaki research group at Nippon Medical School, Japan. This work was supported in part by the Internaitonal Collaborations in Chemistry Grant from JSPS and NSF, the Nagase Science and Technology Foundation Research Grant, and JSPS grants-in-aid.
    Catalog# Strain name Genotype Amino acid requirement and/or labeling
    RDB15927 ML2 cyo::kan ilvE; Ile, Leu.
    RDB15928 ML3 cyo::kan hisG His.
    RDB15929 ML6 cyo::kan ilvE avtA Ile, Leu, Val.
    RDB15930 ML8 cyo::kan argH Arg.
    RDB15931 ML12 cyo::kan ilvE avtA aspC (Ala), Ile, Leu, Tyr, Val.
    RDB15932 ML14 tyrA (Tyr).
    RDB15933 ML17 glnA Gln.
    RDB15934 ML21 tyrA hisG (Tyr), His.
    RDB15935 ML24 cyo ilvE avtA aspC hisG asnA (Ala), Ile, Leu, Tyr, Val, His.
    RDB15936 ML25 cyo ilvE avtA aspC hisG asnA asnB (Ala), Ile, Leu, Tyr, Val, His, Asn.
    RDB15937 ML26 cyo ilvE avtA aspC hisG argH (Ala), Ile, Leu, Tyr, Val, His, Arg.
    RDB15938 ML31 cyo ilvE avtA aspC hisG argH metA (Ala), Ile, Leu, Tyr, Val, His, Arg, Met.
    RDB15939 ML36 cyo ilvE avtA aspC hisG metA (Ala), Ile, Leu, Tyr, Val, His, Met.
    RDB15940 ML40 cyo ilvE avtA aspC hisG argH metA lysA (Ala), Ile, Leu, Tyr, Val, His, Arg, Met, Lys.
    RDB15941 ML42 cyo ilvE avtA aspC hisG argH metA lysA thrC asnB (Ala), Ile, Leu, Tyr, Val, His, Arg, Met, Lys, Thr.
    RDB15942 ML43 cyo ilvE avtA aspC hisG argH metA lysA thrC asnA asnB (Ala), Ile, Leu, Tyr, Val, His, Arg, Met, Lys, Thr, Asn.
    RDB15943 YM138 cysE Cys.
    RDB15944 MS1 cysE hisG Cys, His.
    RDB15952 RF11 metA Met.
    RDB15945 RF1 glyA Gly.
    RDB15946 RF2 thrC Thr.
    RDB15947 RF4 aspC tyrB Asp, Tyr, (Phe).
    RDB15948 RF5 aspC tyrB hisG Asp, Tyr, (Phe), His.
    RDB15949 RF6 proC Pro.
    RDB15950 RF8 asnA asnB Asn.
    RDB15951 RF10 lysA Lys.
    RDB15953 RF12 trpA trpB Trp.
    RDB15954 RF13 aspC tyrB trpA trpB Asp, Tyr, Trp, (Phe).
    RDB15955 RF15 aspC tyrB trpA trpB glyA serB Asp, Tyr, Trp, (Phe), Gly, Ser.
    RDB15956 RF16 aspC tyrB trpA trpB glyA serB cysE Asp, Tyr, Trp, (Phe), Gly, Ser, Cys, Ala.
    RDB15957 RF17 aspC tyrB ilvE Asp, Tyr, Phe, Ile, Leu.
    RDB15958 RF18 aspC tyrB ilvE avtA Asp, Tyr, Phe, Ile, Leu, Val.
    RDB15959 RF21 aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC) Asp, Tyr, Phe, Ile, Leu, Val.
    RDB15960 EH1 thrC ilvA Thr, Ile.
  • Please refer web site of Escherichia coli auxotrophic expression strains (by the depositor), Lin et al., Methods 55 (4): 370-378 (2011), Iwasaki et al., J. Am. Chem. Soc. 134 (48): 19731-19738 (2012), and/or Lin et al., Methods Enzymol. 565: 45-66 (2015).


Escherichia coli host and vectors

  • Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium.
    Inouye, S., Sahara, Y.
    Protein Expr. Purif. 53 (2): 384-389 (2007). PMID 17275329.

    Catalog# Resource name Description
    RDB19856 piP-H-XEL Bacterial expression vector. Secretion vector with OmpA signal peptide sequence and His-Tag under the control of Ipp promoter in E. coli.
  • Molecular engineering of the salicylate-inducible transcription factor Sal7AR for orthogonal and high gene expression in Escherichia coli.
    Miyazaki, K.
    PLoS One 13 (4): e0194090 (2018). PubMed PMID 29641575.

    Catalog# Resource name Description
    RDB17946 pSAL7A35VS Bacterial expression vector for the salicylate-inducible high expression system in E. coli.
  • Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.
    Ihara, K., Sato, K., Hori, H., Makino, Y., Shigenobu, S., Ando, T., Isogai, E., Yoneyama, H.
    J. Biosci. Bioeng. 123 (4): 444-450 (2017). PubMed PMID 28057466.

    Catalog# Resource name Description
    RDB15921 MG1655 delta lrp Escherichia coli host strain. MG1655 with a deletion in lrp gene.
    RDB15922 MG1655 delta lacZ Escherichia coli host strain. MG1655 with a deletion in lacZ.
    RDB15923 MG1655 delta lacZ delta lrp Escherichia coli host strain. MG1655 with deletion in lrp and lacZ genes
    RDB15924 pQE-Lrp Expression vector of Escherichia coli K-12 MG1655 leucine-responsive regulatory protein (Lrp).
  • Improvement of (R)-1,3-butanediol production by engineered Escherichia coli.
    Kataoka, N., Vangnai, A.S., Tajima, T., Nakashimada, Y., Kato, J.
    J. Biosci. Bioeng. 115(5): 475-480 (2013). PubMed PMID 23290993.

    Catalog num. Resource name Description
    RDB15466 pNK E. coli expression vector, PA1lacO-1 promoter, rrnBT1T2 terminator, p15A and pAM alpha1 origin.
  • MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates.
    Fukunaga, R., Hunter, T.
    EMBO J., 16, 1921-1933 (1997). PubMed PMID 9155018.

    Catalog num. Resource name Description
    RDB01911 Lambda GEX5 Lambda phage cloning vector producing GST fusion protein.
  • Phosphorylation of two eukaryotic transcription factors, Jun dimerization protein 2 and activation transcription factor 2, in Escherichia coli by Jun N-terminal kinase 1.
    Murata, T., Shinozuka, Y., Obata, Y., Yokoyama, K.K.
    Anal. Biochem., 376, 115-121 (2008). PubMed PMID 18307971.

    Catalog num. Resource name Description
    RDB06211 pT7Tet1 Expression vector, bacterial.

  • Other vectors
    Catalog num. Resource name ori Registance (E. coli) Cloning Promoter Tag
    Amp Tet Cm lac T7 FLAG His GST
    RDB06084 pETEG5 pBR322 Restriction Enzyme
    RDB06211 pT7Tet1 p15A Restriction Enzyme
    RDB06212 pT7Tet6 p15A Restriction Enzyme
    RDB07041 pETHFFc pBR322 Gateway®
    RDB07050 pETF pBR322 Restriction Enzyme
    RDB07051 pT7FCam p15A Restriction Enzyme

Bifidobacterium longum bacterial host and vectors

  • Construction of Escherichia coli-Bifidobacterium longum shuttle vector transforming B. longum 105-A and 108-A.
    Matsumura, H., Takeuchi, A., Kano, Y.
    Biosci. Biotechnol. Biochem. 61 (7): 1211-1212 (1997). PubMed PMID 9255988.

    Catalog num. Resource name Description
    JCM 31944 105-A Host strain of Bifidobacterium longum 105-A available from JCM.
  • Development of a double-crossover markerless gene deletion system in Bifidobacterium longum: functional analysis of the α-galactosidase gene for raffinose assimilation.
    Hirayama, Y., Sakanaka, M., Fukuma, H., Murayama, H., Kano, Y., Fukiya, S., Yokota, A.
    Appl. Environ. Microbiol. 78 (14): 4984-4994 (2012). PubMed PMID 22582061.

    Catalog num. Resource name Description
    RDB15972 pTBR101-CM Bifidobacterium longum shuttle vector
    RDB15973 pBS423 delta repA Bifidobacterium longum shuttle vector
    RDB15974 pBS423 Bifidobacterium longum shuttle vector

Bacillus stearothermophilus bacterial host and vectors

  • A newly isolated Bacillus stearothermophilus K1041 and its transformation by electroporation.
    Narumi, I., Sawakami, K., Nakamoto, S., Nakayama, N., Yanagisawa, T., Takahashi, N., Kihara, H.
    Biotechnol. Tech. 6, 83-86 (1992).

    Catalog num. Resource name Description
    RDB00139 K1041 Host strain of Bacillus stearothermophilus for pSTE33.
  • Nakayama, N., A new shuttle vector for Bacillus stearothermophilus and Escherichia coli.
    Nakayama, N., Narumi, I., Nakamoto, S., Kihara, H.
    Biotechnol. letters, 14, 649-652 (1992).

    Catalog num. Resource name Description
    RDB00919 pSTE12 Bacillus stearothermophilus shuttle vector
  • Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus, Baciluss subtilis, and Escherichia coli.
    Narumi, I., Nakayama, N., Makamoto, S., Kimura, T., Yanagisawa, T., Kihara, H.
    Biotechnol. letters, 15, 815-820 (1993).

    Catalog num. Resource name Description
    RDB00920 pSTE33 Bacillus stearothermophilus shuttle vector
  • DNA resources as well as Bacillus subtilis strains [link] are available at “Bacillus subtilis, National BioResource Project”, National Institute of Genetics.

(GRP0028e 2018.06.01 T.M.)


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