IGEM:Harvard/2006/Fusion proteins

Protein domain BioBricks presentation

Cell surface targeting

A suggestion: although it might be bad form, when citing an article, just refer to the PMID. This is more useful than the pretty pubmed link which isn't smart enough to know when you are logged in with a HUID.

Plan

See the slides for some brief info.

Express monomeric streptavidin on E. coli cell surface

Scaffold 1: GPI anchor

We've looked at this less so far since autotransporter seemed more attractive, but maybe we should look at it more.

Scaffold 2: Beta autotransporter

See some of the readings below for more info.

Major questions/concerns

1. Where can we get the DNA expressing the scaffold protein. I need to look more into this, but I don't think we can just order it.
2. Where do we get streptavidin DNA (smaller problem)
3. What constraints do we have to worry about with streptavidin? In the original and later autotransporter papers, the passenger protein was mutated to lack cysteines in order to prevent disulfide bond formation in the periplasm: tertiary structure prevents passage of the protein through the beta barrel.
4. How much time to get a first experiment up and running? This depends on how much work we have to do with the scaffold protein sequence and how fast we can get them (likely a long time).
5. Will we be able to correspond with experts on this system? The founder of autodisplay, Joachim Jose, has not responded to some intial questions about autotransporter.

Demonstrate control of a nucleic acid interface between proteins and the cell surface

Notes

(Maybe Un-, but more likely..) fortunately, another group has done work like this. We'll expand on the "adaptamer" concept of Tahiri-Alaoui et al. (12000850). In this paper, the group evolved one aptamer which bound streptavidin, then spliced it to a CopA RNA. Then they spliced a complementary CopT RNA to a CD4 aptamer. Following CopA/CopT binding, the resulting construct could bind both CD4 and streptavidin. Neato. CopA and CopT bind each other by a kissing complex between two loops; mutations have been introduced before, so looking at those studies might be a good starting places for adjusting the dissociation rate (16199086).

The reason they go through the trouble of using CopA and CopT is that they maintain stable secondary structures; the group reports that when you just have any old complementary strands, the binding affinities of the aptamers goes down considerably. We may be able to design our own RNA-RNA interfaces to have well-defined secondary structure, enhancing our ability to control dissociation rate.

Additionally, we want to make one of these aptamers bind to the E. coli cell surface. There is a serious dearth of known aptamers that do this. The only report about one is 15541352; their aptamer binds LPS, a major component of the E. coli outer membrane, so it has a lot of targets. They don't give a Kd value, maybe because it didn't matter too much with so much LPS. Hope we can get our hands on the sequence..

One thing to consider: in the absence of aptamers that bind to the cell surface, we might as well try it with aptamers that bind proteins in vitro as done in 12000850. Why worry about cells for now? We're interested in a proof of principle.

Major Questions/concerns

1. What aptamers exist that bind the E. coli cell surface??? (and have public sequences! The sequence of the aptamer that binds LPS is not reported in the paper.)
2. Is there any chance that we'll be able to evolve our own aptamer this summer? Online resources point toward 'no' but see last question.
3. What other secondary structures might work as interfaces? If we design our own, the online programs mfold and Vienna RNA may be useful. Otherwise, we'll have to go with known complementary structures. Do the faculty members have any suggestions for structures?
4. Time and reliability: Hopefully short! Aptamer sequences are relatively short, so we'll likely be able to order them instead of asking groups for them. Then we'd try tons of intermediate interfaces, which is just a matter of ordering more oligos.
5. Will people respond to email? I've emailed David Liu about evolving aptamers and Sulatha Dwarakanath (author of 15541352) about aptamers that target the E. coli cell surface to no response. It would be quite helpful to hear their thoughts.

Other projects

A couple of related projects that we haven't really researched..

1. Having E. coli export an aptamer outside the cell
2. Using aptamers to actually stimulate a response on the cell surface

Reading

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 |
12. Samuelson P, Gunneriusson E, Nygren PA, and Ståhl S. Display of proteins on bacteria. J Biotechnol. 2002 Jun 26;96(2):129-54. DOI:10.1016/s0168-1656(02)00043-3 |

13. pmid=

Notes on Autodisplay review (display1) 16369779

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) 11024362

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) 9624691

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.

More bacterial display (display4) 12039531

This review's gram-negative bacteria section is slightly more recent than display1 but contains almost no more information. It does have more explicit weight constraints on the proteins you can display for each system and pays slightly more attention to other constraints, but nothing that interesting. It has an equally comprehensive section on gram-positive bacteria. You might as well look at this one and not display1, though because it was published in 2002 vs. 2000 for display1.

It does mention that gram-positive bacteria are more suitable for display. Tested gram-postive display systems are better at displaying large proteins. Additionally, protein only need pass through one membrane versus moving through the cytoplasmic membrane and achieving the correct orientation in the outer membrane. Finally, gram-positive bacteria are more durable and easier to test under harsh lab procedures.

Aptamers

14. Dwarakanath S, Bruno JG, Shastry A, Phillips T, John AA, Kumar A, and Stephenson LD. Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria. Biochem Biophys Res Commun. 2004 Dec 17;325(3):739-43. DOI:10.1016/j.bbrc.2004.10.099 |
15. Bruno JG and Kiel JL. Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods. Biotechniques. 2002 Jan;32(1):178-80, 182-3. DOI:10.2144/02321dd04 |
16. 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 |
17. Nordström K. Plasmid R1--replication and its control. Plasmid. 2006 Jan;55(1):1-26. DOI:10.1016/j.plasmid.2005.07.002 |
18. Kolb FA, Malmgren C, Westhof E, Ehresmann C, Ehresmann B, Wagner EG, and Romby P. An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment. RNA. 2000 Mar;6(3):311-24. DOI:10.1017/s135583820099215x |

Quantum dots conjugated to aptamers (apt1) 15541352

This was a neat study in which quantum dots were conjugated to antibodies or aptamers that target E. coli cell surfaces. The point of the article is that the emissions from the quantum dots changed following binding, but what's relevant to us is the aptamer they used. They evolved it on their own using the method of apt2 (probably out of convenience: both were Austin-based companies). The aptamer recognizes lipopolysaccharide (LPS O111:B4). I am currently emailing to find out why they chose LPS. At least we know of one aptamer that binds the E. coli cell surface...

Adaptamers (apt3) 12000850

This paper is also listed in the streptavidin section. See "Notes" under "Demonstrate Control.." project for a description.

CopA/CopT (apt4) 16199086

This recent review paper has a description of the CopA/CopT interaction which is utilized in apt3.

CopA/CopT (apt5) 10744017

This paper reveals the extremely intricate "deep-kissing" structure formed between CopA and CopT.

Streptavidin

19. Bayer EA and Wilchek M. Avidin- and streptavidin-containing probes. Methods Enzymol. 1990;184:174-87. DOI:10.1016/0076-6879(90)84272-i |
20. 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 |
21. 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.
22. 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 |
23. 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 |
24. 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 |
25. 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 |
26. 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 |
27. 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 |
28. 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 |
29. 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 |
30. 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 |
31. 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 |
32. 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 |
33. 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 |
34. Lemercier G and Johnsson K. Chimeric streptavidins with reduced valencies. Nat Methods. 2006 Apr;3(4):247-8. DOI:10.1038/nmeth0406-247 |
35. 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 |
36. 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 |
37. 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 |
38. 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 |
39. 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 |
40. 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 |
41. 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 |
42. 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 |
43. 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 |
44. CHAIET L and WOLF FJ. THE PROPERTIES OF STREPTAVIDIN, A BIOTIN-BINDING PROTEIN PRODUCED BY STREPTOMYCETES. Arch Biochem Biophys. 1964 Jul 20;106:1-5. DOI:10.1016/0003-9861(64)90150-x |

Display of Tetrameric streptavidin on B. subtilis spore surfaces (sa23) 16289701

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.

ompA

45. Wang JY and Chao YP. Immobilization of cells with surface-displayed chitin-binding domain. Appl Environ Microbiol. 2006 Jan;72(1):927-31. DOI:10.1128/AEM.72.1.927-931.2006 |
46. Georgiou G, Stephens DL, Stathopoulos C, Poetschke HL, Mendenhall J, and Earhart CF. Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions. Protein Eng. 1996 Feb;9(2):239-47. DOI:10.1093/protein/9.2.239 |
47. Stathopoulos C, Georgiou G, and Earhart CF. Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane. Appl Microbiol Biotechnol. 1996 Mar;45(1-2):112-9. DOI:10.1007/s002530050657 |

Related articles

48. 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) 9893944

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.

Working Team Members

• Perry Tsai (talk)
• Lewis Hahn (talk)