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SHuffle® publications

Protien Expression Product Hero

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Review on protein expression, mentioning SHuffle:

[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]

SHuffle characterization: [11], [12]

Protein expression in a fermenter using SHuffle: [13]

Protocol describing the use of SHuffle in protein expression: [14]

Expression of proteins in SHuffle:

Substrate #cys Yield (mg/L) Year Ref
Herpes virus membrane glycoprotein 2 1-2 2011 [15]
Pregnancy Associated Malaria component (Var2CSA) 11 0.1-20 2011 [16]
VEGF 6 - 2012 [17]
Gluc 10 2 2012 [18]
VHH (nanobody) 2 1-10 periplasm 2013 [19]
~100 SHuffle
VHH (nanobody) 2 200 2013 [20]
Lysozyme 8 30 (X3000 improvement) 2013 [21]
Neurotoxin 6 1 2013 [22]
Cu,Zn-superoxidedismutase 2 20 2013 [23]
Trx-Thionin 8 1-2 2013 [24]
extracellular domain of interleukin-23 receptor 10 - 2013 [25]
Fibroblast growth factor 4 0.5-2 2014 [26]
P. falciparum surface protein 12 23 2014 [27]
Fab 4 0.3-1 2014 [28]
Limonene epoxide hydrolase 2 5-10 2014 [29]
FtsN membrane protein 2 2 2014 [30]
Human and murine anaphylatoxins 6 25 2014 [31]
Chicken Follistatin 36 6 (failed in periplasm) 2014 [32]
Barley Subtilase 10 0.4 2014 [33]
Barley Hordein 8~10 Purified from Inclusion body 2014 [34]
Mdm2-enterokinase   - 2014 [35]
β-lactamase-inhibitor fusion 2 - 2014 [36]
human kringle domain-GFP fusion 6 7-20 2014 [37]
Lipase 10 - 2014 [38]
(NF)- kB ligand 4 52 2014 [39]
IgG single chain with variable domains 4 - 2014 [40]
GFP nanobody-PHB beads 2 9~16 mg/g beads (>>BL21) 2014 [41]
IgaA periplasmic domain 4 - 2014 [42]
DNA primase-polymerase 14 (cytoplasmic protein) 2015 [43]
Phosphodiesterase 4 0.2 2015 [44]
IgG 16 1-20 2015 [45]
scFv 8 - 2015 [46]
Vibrio cholerae cytolysin - - 2015 [47]
Cyclohexanone monooxygenase - - 2015 [48]
Firefly Luciferase - - 2015 [49]
FtsN - - 2015 [50]
Phobalysin - - 2015 [51]
Chicken Follistatin 36 7 – 20 2015 [52]
Phosphatidic acid transferase Mdm35 4 - 2015 [53]
scFv 4 - 2015 [54]
Lectin-like 2 - 2015 [55]
gamma-B crystallin 7 - 2016 [56]
Endogluconase - - 2016 [57]
Lectin 0 115 2016 [58]
VEGF 8 1-2 2016 [59]


  1. Berkmen M: Production of disulfide-bonded proteins in Escherichia coli. Protein Expr Purif 2012, 82(1):240-251.
  2. de Marco A: Recent contributions in the field of the recombinant expression of disulfide bonded proteins in bacteria. Microbial cell factories 2012, 11:129.
  3. Berkmen M: Oxidative Folding of Proteins in Escherichia Coli. BioPharm International 2012, 25(5):48-54
  4. Salinas G, Pellizza L, Margenat M, Flo M, Fernandez C: Tuned Escherichia coli as a host for the expression of disulfide-rich proteins. Biotechnol J 2011, 6(6):686-699.
  5. Ritter A: Next-Gen Expression Systems. Pharmaceutical Technology 2011, 35(6):36-39.
  6. Zhang L, Chou CP, Moo-Young M: Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system. Biotechnol Adv 2011, 29(6):923-929.
  7. Costa S, Almeida A, Castro A, Domingues L: Fusion tags for protein solubility, purification and immunogenicity in : the novel Fh8 system. Frontiers in microbiology 2014, 5:63.
  8. Lebendiker M, Danieli T: Production of prone-to-aggregate proteins. FEBS letters 2014, 588(2):236-246.
  9. Rosano GL, Ceccarelli EA: Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology 2014, 5:172.
  10. de Marco A: Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs. Microbial cell factories 2015, 14:125.
  11. Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M: SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microbial cell factories 2012, 11(1):56.
  12. Nozach H, Fruchart-Gaillard C, Fenaille F, Beau F, Ramos OH, Douzi B, Saez NJ, Moutiez M, Servent D, Gondry M et al: High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli. Microbial cell factories 2013, 12:37.
  13. Samuelson JC, Causey TB, Berkmen M: Disulfide-Bonded Protein Production in E. coli. Genetic Engineering and Biotechnology News 2012, 32(3):35.
  14. Ren G, Ke N, Berkmen M: Use of the SHuffle Strains in Production of Proteins. Curr Protoc Protein Sci 2016, 85:5 26 21-25 26 21.
  15. Tait AR, Straus SK: Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield. Microbial cell factories 2011, 10:51.
  16. Srivastava A, Gangnard S, Dechavanne S, Amirat F, Lewit Bentley A, Bentley GA, Gamain B: Var2CSA minimal CSA binding region is located within the N-terminal region. PloS one 2011, 6(5):e20270.
  17. Knapp RT, Wu CH, Mobilia KC, Joester D: Recombinant sea urchin vascular endothelial growth factor directs single-crystal growth and branching in vitro. Journal of the American Chemical Society 2012, 134(43):17908-17911.
  18. Tzertzinis G, Schildkraut E, Schildkraut I: Substrate cooperativity in marine luciferases. PloS one 2012, 7(6):e40099.
  19. Habib I, Smolarek D, Hattab C, Grodecka M, Hassanzadeh-Ghassabeh G, Muyldermans S, Sagan S, Gutierrez C, Laperche S, Le-Van-Kim C et al: V(H)H (nanobody) directed against human glycophorin A: a tool for autologous red cell agglutination assays. Analytical biochemistry 2013, 438(1):82-89.
  20. Zarschler K, Witecy S, Kapplusch F, Foerster C, Stephan H: High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli. Microbial cell factories 2013, 12:97.
  21. Lamppa JW, Tanyos SA, Griswold KE: Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme. J Biotechnol 2013, 164(1):1-8.
  22. Sermadiras I, Revell J, Linley JE, Sandercock A, Ravn P: Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV. PloS one 2013, 8(12):e83202.
  23. Furukawa Y: Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis. Front Cell Neurosci 2013, 7:240.
  24. Abbas A, Plattner S, Shah KH, Bohlmann H: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli. Biotechnology letters 2013, 35(7):1085-1091.
  25. . Kuchar M, Vankova L, Petrokova H, Cerny J, Osicka R, Pelak O, Sipova H, Schneider B, Homola J, Sebo P et al: Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells. Proteins 2014, 82(6):975-989.
  26. Kong B, Guo GL: Soluble Expression of Disulfide Bond Containing Proteins FGF15 and FGF19 in the Cytoplasm of Escherichia coli. PloS one 2014, 9(1):e85890.
  27. Gupta PK, Mukherjee P, Dhawan S, Pandey AK, Mazumdar S, Gaur D, Jain SK, Chauhan VS: Production and Preclinical Evaluation of Plasmodium falciparum MSP-119 and MSP-311 Chimeric Protein, PfMSP-Fu24. Clinical and vaccine immunology : CVI 2014, 21(6):886-897.
  28. Abe R, Jeong HJ, Arakawa D, Dong J, Ohashi H, Kaigome R, Saiki F, Yamane K, Takagi H, Ueda H: Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence. Scientific reports 2014, 4:4640.
  29. Wijma HJ, Floor RJ, Jekel PA, Baker D, Marrink SJ, Janssen DB: Computationally designed libraries for rapid enzyme stabilization. Protein engineering, design & selection : PEDS 2014, 27(2):49-58.
  30. Duncan TR, Yahashiri A, Arends SJ, Popham DL, Weiss DS: Identification of SPOR domain amino acids important for septal localization, peptidoglycan binding, and a disulfide bond in the cell division protein FtsN. Journal of bacteriology 2013, 195(23):5308-5315.
  31. Schatz-Jakobsen JA, Yatime L, Larsen C, Petersen SV, Klos A, Andersen GR: Structural and functional characterization of human and murine C5a anaphylatoxins. Acta Crystallogr D Biol Crystallogr 2014, 70(Pt 6):1704-1717.
  32. Lee SB, Choi R, Park SK, Kim YS: Production of bioactive chicken follistatin315 in Escherichia coli. Appl Microbiol Biotechnol 2014, 98(24):10041-10051.
  33. Plattner S, Gruber C, Altmann F, Bohlmann H: Self-processing of a barley subtilase expressed in E. coli. Protein Expr Purif 2014, 101:76-83.
  34. Rosenkilde AL, Dionisio G, Holm PB, Brinch-Pedersen H: Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins. Phytochemistry 2014, 97:11-19.
  35. Nirantar SR, Li X, Siau JW, Ghadessy FJ: Rapid screening of protein-protein interaction inhibitors using the protease exclusion assay. Biosens Bioelectron 2014, 56:250-257.
  36. Lee HC, Portnoff AD, Rocco MA, DeLisa MP: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism. Scientific reports 2014, 4:7570.
  37. Jeong GM, Kim YS, Jeong KJ: A human kringle domain-based fluorescence-linked immunosorbent assay system. Analytical biochemistry 2014, 451:63-68.
  38. Nars G, Saurel O, Bordes F, Saves I, Remaud-Simeon M, Andre I, Milon A, Marty A: Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems. Protein Expr Purif 2014, 101:14-20.
  39. Maharjan S, Singh B, Bok JD, Kim JI, Jiang T, Cho CS, Kang SK, Choi YJ: Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli. PloS one 2014, 9(5):e96259.
  40. Ta DT, Redeker ES, Billen B, Reekmans G, Sikulu J, Noben JP, Guedens W, Adriaensens P: An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation. Protein engineering, design & selection : PEDS 2015, 28(10):351-363.
  41. Hay ID, Du J, Burr N, Rehm BH: Bioengineering of bacteria to assemble custom-made polyester affinity resins. Appl Environ Microbiol 2015, 81(1):282-291.
  42. Cho SH, Szewczyk J, Pesavento C, Zietek M, Banzhaf M, Roszczenko P, Asmar A, Laloux G, Hov AK, Leverrier P et al: Detecting envelope stress by monitoring beta-barrel assembly. Cell 2014, 159(7):1652-1664.
  43. Guilliam TA, Jozwiakowski SK, Ehlinger A, Barnes RP, Rudd SG, Bailey LJ, Skehel JM, Eckert KA, Chazin WJ, Doherty AJ: Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins. Nucleic Acids Res 2015, 43(2):1056-1068.
  44. Mineta S, Murayama K, Sugimori D: Characterization of glycerophosphoethanolamine ethanolaminephosphodiesterase from Streptomyces sanglieri. J Biosci Bioeng 2015, 119(2):123-130.
  45. Robinson MP, Ke N, Lobstein J, Peterson C, Szkodny A, Mansell TJ, Tuckey C, Riggs PD, Colussi PA, Noren CJ et al: Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nature communications 2015, 6:8072.
  46. Ojima-Kato T, Hashimura D, Kojima T, Minabe S, Nakano H: In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems. J Immunol Methods 2015, 427:58-65.
  47. De S, Bubnys A, Alonzo F, 3rd, Hyun J, Lary JW, Cole JL, Torres VJ, Olson R: The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin. The Journal of biological chemistry 2015, 290(47):28402-28415.
  48. Schmidt S, Genz M, Balke K, Bornscheuer UT: The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase. J Biotechnol 2015, 214:199-211.
  49. Kurihara M, Ohmuro-Matsuyama Y, Ayabe K, Yamashita T, Yamaji H, Ueda H: Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions. Biotechnol J 2015.
  50. Yahashiri A, Jorgenson MA, Weiss DS: Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides. Proceedings of the National Academy of Sciences of the United States of America 2015, 112(36):11347-11352.
  51. Rivas AJ, von Hoven G, Neukirch C, Meyenburg M, Qin Q, Fuser S, Boller K, Lemos ML, Osorio CR, Husmann M: Phobalysin, a Small beta-Pore-Forming Toxin of Photobacterium damselae subsp. damselae. Infect Immun 2015, 83(11):4335-4348.
  52. Lee SB, Park SK, Kim YS: Production of bioactive chicken (Gallus gallus) follistatin-type proteins in E. coli. AMB Express 2015, 5(1):142.
  53. Watanabe Y, Tamura Y, Kawano S, Endo T: Structural and mechanistic insights into phospholipid transfer by Ups1-Mdm35 in mitochondria. Nature communications 2015, 6:7922.
  54. Horwacik I, Golik P, Grudnik P, Kolinski M, Zdzalik M, Rokita H, Dubin G: Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody. Mol Cell Proteomics 2015, 14(10):2577-2590.
  55. Diderrich R, Kock M, Maestre-Reyna M, Keller P, Steuber H, Rupp S, Essen LO, Mosch HU: Structural Hot Spots Determine Functional Diversity of the Candida glabrata Epithelial Adhesin Family. The Journal of biological chemistry 2015, 290(32):19597-19613.
  56. Buhr F, Jha S, Thommen M, Mittelstaet J, Kutz F, Schwalbe H, Rodnina MV, Komar AA: Synonymous Codons Direct Cotranslational Folding toward Different Protein Conformations. Mol Cell 2016, 61(3):341-351.
  57. Crouch LI, Labourel A, Walton PH, Davies GJ, Gilbert HJ: The contribution of non-catalytic carbohydrate binding modules to the activity lytic polysaccharide monooxygenases. The Journal of biological chemistry 2016.
  58. Hirayama M, Shibata H, Imamura K, Sakaguchi T, Hori K: High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120. Mar Biotechnol (NY) 2016, 18(1):144-160.
  59. Nguyen MT, Krupa M, Koo BK, Song JA, Vu TT, Do BH, Nguyen AN, Seo T, Yoo J, Jeong B et al: Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. PloS one 2016, 11(5):e0156296.

Learn about SHuffle® strains for the expression of multi-disulfide bonded and difficult-to-express proteins