IGEM:Harvard/2006/Fusion proteins

Protein domain BioBricks presentation

Cell surface targeting

General Fusion

1. Klein BK, Hill SR, Devine CS, Rowold E, Smith CE, Galosy S, and Olins PO. Secretion of active bovine somatotropin in Escherichia coli. Biotechnology (N Y). 1991 Sep;9(9):869-72. DOI:10.1038/nbt0991-869 |

2. We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.

3. Utsumi R, Brissette RE, Rampersaud A, Forst SA, Oosawa K, and Inouye M. Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate. Science. 1989 Sep 15;245(4923):1246-9. DOI:10.1126/science.2476847 |
4. Moe GR, Bollag GE, and Koshland DE Jr. Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5683-7. DOI:10.1073/pnas.86.15.5683 |
5. Coulton JW, Mason P, Cameron DR, Carmel G, Jean R, and Rode HN. Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):181-92. DOI:10.1128/jb.165.1.181-192.1986 |
6. Sierke SL and Koland JG. SH2 domain proteins as high-affinity receptor tyrosine kinase substrates. Biochemistry. 1993 Sep 28;32(38):10102-8. DOI:10.1021/bi00089a028 |
7. Nordlund HR, Hytönen VP, Laitinen OH, and Kulomaa MS. Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum. J Biol Chem. 2005 Apr 8;280(14):13250-5. DOI:10.1074/jbc.M414336200 |

8. Bradyrhizobium japonicum is an important nitrogenfixing symbiotic bacterium, which can form root nodules on soybeans. These bacteria have a gene encoding a putative avidin- and streptavidin-like protein, which bears an amino acid sequence identity of only about 30% over the core regions with both of them. We produced this protein in Escherichia coli both as the full-length wild type and as a C-terminally truncated core form and showed that it is indeed a high affinity biotin-binding protein that resembles (strept)avidin structurally and functionally. Because of the considerable dissimilarity in the amino acid sequence, however, it is immunologically very different, and polyclonal rabbit and human antibodies toward (strept)avidin did not show significant cross-reactivity with it. Therefore this new avidin, named bradavidin, facilitates medical treatments such as targeted drug delivery, gene therapy, and imaging by offering an alternative tool for use if (strept)avidin cannot be used, because of a deleterious patient immune response for example. In addition to its medical value, bradavidin can be used both in other applications of avidin-biotin technology and as a source of new ideas when creating engineered (strept)avidin forms by changing or combining the desired parts, interface patterns, or specific residues within the avidin protein family. Moreover, the unexpected discovery of bradavidin indicates that the group of new and undiscovered bacterial avidin-like proteins may be both more diverse and more common than hitherto thought.

E. coli cell surface display

9. Jose J. Autodisplay: efficient bacterial surface display of recombinant proteins. Appl Microbiol Biotechnol. 2006 Feb;69(6):607-14. DOI:10.1007/s00253-005-0227-z |
10. Cornelis P. Expressing genes in different Escherichia coli compartments. Curr Opin Biotechnol. 2000 Oct;11(5):450-4. DOI:10.1016/s0958-1669(00)00131-2 |
11. Jung HC, Lebeault JM, and Pan JG. Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nat Biotechnol. 1998 Jun;16(6):576-80. DOI:10.1038/nbt0698-576 |

Notes on Autodisplay review (display1)

This is a review about the Autodisplay system used to express proteins on the E. coli cell surface. Some of the important features of this system are:

• Utilizes E.coli-native AIDA-I as scaffold
• Detection: monoclonal antibodies and protease cleavage sites created
• Protein unfolded during transport to cell surface.
• Variety of proteins have been displayed (p.610)
• Possibly to display catalytically active enzymes.
• Dimerization of proteins has been observed! (unique to this system); work on tetramers in progress. Protein anchor floats around membrane.
• Many proteins displayed: ~10^5 without loss of cell viability

The writer claims that the system is superior compared to all other display systems, but he might be biased since he created it.

Notes on expressing genes in different compartments review (display2)

This is a more general review which is actually mostly about cell-surface display techniques. It offers a wider range of options, but came out in 2000, so new options probably have become available.

• 5 options; a good summary is found on page 3.
  • Porins- Insert protein <= 60 residues
  • Fimbriae- Insert protein <= 15 residues
  • Lipoproteins
  • GPI anchor
  • Beta-autotransporter - same system as that used in display1

• The latter three were claimed to support large polypeptides. However, looking at a few of the articles seemed to suggest that they actually only tried them out with small polypeptides. The winners appear to be GPI anchors and beta autotransporters, although this might have changed in recent years. See display3 for the use of GPI anchors.

GPI anchor(display3)

I didn't read this one that thoroughly, but the point is that they forced E. coli to express functional levansucrase bound to a GPI anchor, Inp. Using these cells, they successfully converted sucrose to levan, which seems neat. Levansucrase, is about 400 a.a. residues long.

Streptavidin

13. Bayer EA and Wilchek M. Avidin- and streptavidin-containing probes. Methods Enzymol. 1990;184:174-87. DOI:10.1016/0076-6879(90)84272-i |
14. Sano T and Cantor CR. Expression of a cloned streptavidin gene in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jan;87(1):142-6. DOI:10.1073/pnas.87.1.142 |
15. Sano T and Cantor CR. Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea. J Biol Chem. 1990 Feb 25;265(6):3369-73.
16. Sano T and Cantor CR. Expression vectors for streptavidin-containing chimeric proteins. Biochem Biophys Res Commun. 1991 Apr 30;176(2):571-7. DOI:10.1016/s0006-291x(05)80222-0 |
17. Sano T, Pandori MW, Chen X, Smith CL, and Cantor CR. Recombinant core streptavidins. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules. J Biol Chem. 1995 Nov 24;270(47):28204-9. DOI:10.1074/jbc.270.47.28204 |
18. Sano T, Smith CL, and Cantor CR. Expression and purification of recombinant streptavidin-containing chimeric proteins. Methods Mol Biol. 1997;63:119-28. DOI:10.1385/0-89603-481-X:119 |
19. Szafranski P, Mello CM, Sano T, Smith CL, Kaplan DL, and Cantor CR. A new approach for containment of microorganisms: dual control of streptavidin expression by antisense RNA and the T7 transcription system. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1059-63. DOI:10.1073/pnas.94.4.1059 |
20. Sano T, Vajda S, Smith CL, and Cantor CR. Engineering subunit association of multisubunit proteins: a dimeric streptavidin. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6153-8. DOI:10.1073/pnas.94.12.6153 |
21. Kaplan DL, Mello C, Sano T, Cantor C, and Smith C. Streptavidin-based containment systems for genetically engineered microorganisms. Biomol Eng. 1999 Dec 31;16(1-4):135-40. DOI:10.1016/s1050-3862(99)00040-6 |
22. Sano T and Cantor CR. Streptavidin-containing chimeric proteins: design and production. Methods Enzymol. 2000;326:305-11. DOI:10.1016/s0076-6879(00)26061-8 |
23. Farlow SJ, Wang RJ, Pandori MW, and Sano T. A chimera of a gelatinase inhibitor peptide with streptavidin as a bifunctional tumor targeting reagent. FEBS Lett. 2002 Apr 10;516(1-3):197-200. DOI:10.1016/s0014-5793(02)02565-6 |
24. McDevitt TC, Nelson KE, and Stayton PS. Constrained cell recognition peptides engineered into streptavidin. Biotechnol Prog. 1999 May-Jun;15(3):391-6. DOI:10.1021/bp990043n |
25. Farlow SJ, Wang RJ, Pandori MW, and Sano T. A chimera of a gelatinase inhibitor peptide with streptavidin as a bifunctional tumor targeting reagent. FEBS Lett. 2002 Apr 10;516(1-3):197-200. DOI:10.1016/s0014-5793(02)02565-6 |
26. Nordlund HR, Hytönen VP, Laitinen OH, and Kulomaa MS. Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum. J Biol Chem. 2005 Apr 8;280(14):13250-5. DOI:10.1074/jbc.M414336200 |
27. Tahiri-Alaoui A, Frigotto L, Manville N, Ibrahim J, Romby P, and James W. High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands. Nucleic Acids Res. 2002 May 15;30(10):e45. DOI:10.1093/nar/30.10.e45 |
28. Lemercier G and Johnsson K. Chimeric streptavidins with reduced valencies. Nat Methods. 2006 Apr;3(4):247-8. DOI:10.1038/nmeth0406-247 |
29. Wu SC and Wong SL. Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem. 2005 Jun 17;280(24):23225-31. DOI:10.1074/jbc.M501733200 |
30. Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A, and Ting AY. A monovalent streptavidin with a single femtomolar biotin binding site. Nat Methods. 2006 Apr;3(4):267-73. DOI:10.1038/nmeth861 |
31. Qureshi MH and Wong SL. Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins. Protein Expr Purif. 2002 Aug;25(3):409-15. DOI:10.1016/s1046-5928(02)00021-9 |
32. Qureshi MH, Yeung JC, Wu SC, and Wong SL. Development and characterization of a series of soluble tetrameric and monomeric streptavidin muteins with differential biotin binding affinities. J Biol Chem. 2001 Dec 7;276(49):46422-8. DOI:10.1074/jbc.M107398200 |
33. Srisawat C and Engelke DR. Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins. RNA. 2001 Apr;7(4):632-41. DOI:10.1017/s135583820100245x |
34. Aslan FM, Yu Y, Mohr SC, and Cantor CR. Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein. Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8507-12. DOI:10.1073/pnas.0503112102 |
35. Wu SC and Wong SL. Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins. Protein Expr Purif. 2006 Apr;46(2):268-73. DOI:10.1016/j.pep.2005.10.006 |
36. Gallizia A, de Lalla C, Nardone E, Santambrogio P, Brandazza A, Sidoli A, and Arosio P. Production of a soluble and functional recombinant streptavidin in Escherichia coli. Protein Expr Purif. 1998 Nov;14(2):192-6. DOI:10.1006/prep.1998.0930 |
37. Kim JH, Lee CS, and Kim BG. Spore-displayed streptavidin: a live diagnostic tool in biotechnology. Biochem Biophys Res Commun. 2005 May 27;331(1):210-4. DOI:10.1016/j.bbrc.2005.03.144 |

Display of Tetrameric streptavidin on B. subtilis spore surfaces (sa23)

As the title sugggests, a group was actually able to express _tetrameric_ streptavidin on the B. subtilis cell surface. This was a lot easier than doing it in other types of bacteria since they didn't have to worry about getting across the cell membrane or cell wall: when B. subtilis forms spores, a bunch of proteins form in the cytosol which then become the coats of spores... Additionally, spores form approximately when cells stop dividing, which somehow defeats the biotin sequestration problem. Actually, they never do a specific experiment to show this, but we are probably supposed to infer that it was enough to just see the streptavidin on the surface(?) Regardless, this organism is pretty different from E. coli, so it probably isn't terribly important.

Related articles

38. Valls M, González-Duarte R, Atrian S, and De Lorenzo V. Bioaccumulation of heavy metals with protein fusions of metallothionein to bacterial OMPs. Biochimie. 1998 Oct;80(10):855-61. DOI:10.1016/s0300-9084(00)88880-x |

Cd2+ binding

other1 is an old review article I came across talking about expressing metallothioneins on the cell surface of E. coli; these things bind Cd2+, so those interested in pollution cleanup might want to take a look.