Skip to main content
NCBI home page
Wiley - PMC COVID-19 Collection logoLink to Wiley - PMC COVID-19 Collection
. 2009 Feb 9;103(4):696–705. doi: 10.1002/bit.22282

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Turgay Kacar 1,2, Melvin T Zin 1, Christopher So 1, Brandon Wilson 1, Hong Ma 1, Nevin Gul‐Karaguler 2, Alex K‐Y Jen 1,3, Mehmet Sarikaya 1,4, Candan Tamerler 1,2,✉
PMCID: PMC7161797  PMID: 19309754

Abstract

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.

Keywords: inorganic binding peptides, enzymes, oriented‐immobilization, genetic fusion, self‐assembly

References

  1. Adey NB, Mataragnon AH, Rider JE, Carter JM, Kay BK. 1995. Characterization of phage that bind plastic from phage‐displayed random peptide libraries. Gene 156(1): 27–31. [DOI] [PubMed] [Google Scholar]
  2. Anderson RA, Bosron WF, Kennedy FS, Vallee BL. 1975. Role of magnesium in Escherichia‐coli alkaline‐phosphatase. Proc Natl Acad Sci USA 72(8): 2989–2993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Babu VRS, Kumar MA, Karanth NG, Thakur MS. 2004. Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications. Biosens Bioelectron 19(10): 1337–1341. [DOI] [PubMed] [Google Scholar]
  4. Bailey RC, Kwong GA, Radu CG, Witte ON, Heath JR. 2007. DNA‐encoded antibody libraries: A unified platform for multiplexed cell sorting and detection of genes and proteins. J Am Chem Soc 129(7): 1959–1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bornscheuer UT. 2003. Immobilizing enzymes: How to create more suitable biocatalysts. Angew Chem Int Ed 42(29): 3336–3337. [DOI] [PubMed] [Google Scholar]
  6. Bradford MM. 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein‐dye binding. Anal Biochem 72(1–2): 248–254. [DOI] [PubMed] [Google Scholar]
  7. Brennan CA, Christianson K, Lafleur MA, Mandecki W. 1995. A molecular sensor system based on genetically‐engineered alkaline‐phosphatase. Proc Natl Acad Sci USA 92(13): 5783–5787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown S. 1997. Metal‐recognition by repeating polypeptides. Nat Biotechnol 15(3): 269–272. [DOI] [PubMed] [Google Scholar]
  9. Brown S, Sarikaya M, Johnson E. 2000. A genetic analysis of crystal growth. J Mol Biol 299(3): 725–735. [DOI] [PubMed] [Google Scholar]
  10. Dai HX, Choe WS, Thai CK, Sarikaya M, Traxler BA, Baneyx F, Schwartz DT. 2005. Nonequilibrium synthesis and assembly of hybrid inorganic‐protein nanostructures using an engineered DNA binding protein. J Am Chem Soc 127(44): 15637–15643. [DOI] [PubMed] [Google Scholar]
  11. Fairman R, Akerfeldt KS. 2005. Peptides as novel smart materials. Curr Opin Struct Biol 15(4): 453–463. [DOI] [PubMed] [Google Scholar]
  12. Goren M, Galley N, Lennox RB. 2006. Adsorption of alkylthiol‐capped gold nanoparticles onto alkylthiol self‐assembled monolayers: An SPR study. Langmuir 22(3): 1048–1054. [DOI] [PubMed] [Google Scholar]
  13. Holmes TC. 2002. Novel peptide‐based biomaterial scaffolds for tissue engineering. Trends Biotechnol 20(1): 16–21. [DOI] [PubMed] [Google Scholar]
  14. Ishikawa K, Yamada K, Kumagai S, Sano KI, Shiba K, Yamashita I, Kobayashi M. 2008. Adsorption properties of a gold‐binding peptide assessed by its attachment to a recombinant apoferritin molecule. Appl Phys Expr 1(3): 034006‐1–034006‐3. [Google Scholar]
  15. Johnson S, Evans D, Laurenson S, Paul D, Davies AG, Ferrigno PK, Walti C. 2008. Surface‐immobilized peptide aptamers as probe molecules for protein detection. Anal Chem 80(4): 978–983. [DOI] [PubMed] [Google Scholar]
  16. Jung YW, Kang HJ, Lee JM, Jung SO, Yun WS, Chung SJ, Chung BH. 2008. Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide. Anal Biochem 374(1): 99–105. [DOI] [PubMed] [Google Scholar]
  17. Kacar T, Ray J, Gungormus M, Oren EE, Tamerler C, Sarikaya M. 2009. Quartz binding peptides as molecular linkers towards fabricating multifunctional micropatterned substrates. Adv Mater 21: 295–299. [Google Scholar]
  18. Karpovich DS, Blanchard GJ. 1994. Direct measurement of the adsorption‐kinetics of alkanethiolate self‐assembled monolayers on a microcrystalline gold surface. Langmuir 10(9): 3315–3322. [Google Scholar]
  19. Kasemo B. 2002. Biological surface science. Surf Sci 500(1–3): 656–677. [Google Scholar]
  20. Kenan DJ, Walsh EB, Meyers SR, O'Toole GA, Carruthers EG, Lee WK, Zauscher S, Prata CAH, Grinstaff MW. 2006. Peptide‐PEG amphiphiles as cytophobic coatings for mammalian and bacterial cells. Chem Biol 13(7): 695–700. [DOI] [PubMed] [Google Scholar]
  21. Kramer RM, Li C, Carter DC, Stone MO, Naik RR. 2004. Engineered protein cages for nanomaterial synthesis. J Am Chem Soc 126(41): 13282–13286. [DOI] [PubMed] [Google Scholar]
  22. Krauland EM, Peelle BR, Wittrup KD, Belcher AM. 2007. Peptide tags for enhanced cellular and protein adhesion to single‐crystal line sapphire. Biotechnol Bioeng 97(5): 1009–1020. [DOI] [PubMed] [Google Scholar]
  23. Kriplani U, Kay BK. 2005. Selecting peptides for use in nanoscale materials using phagedisplayed combinatorial peptide libraries. Curr Opin Biotechnol 16(4): 470–475. [DOI] [PubMed] [Google Scholar]
  24. Kroger N, Deutzmann R, Sumper M. 2001. Silica‐precipitating peptides from diatoms—The chemical structure of silaffin‐1A from Cylindrotheca fusiformis. J Biol Chem 276(28): 26066–26070. [DOI] [PubMed] [Google Scholar]
  25. Kumada Y, Tokunaga Y, Imanaka H, Imamura K, Sakiyama T, Katoh S, Nakanishi K. 2006. Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins. Biotechnol Prog 22(2): 401–405. [DOI] [PubMed] [Google Scholar]
  26. Kwon Y, Coleman MA, Camarero JA. 2006. Selective immobilization of proteins onto solid supports through split‐intein‐mediated protein trans‐splicing. Angew Chem Int Ed 45(11): 1726–1729. [DOI] [PubMed] [Google Scholar]
  27. Lee KB, Park SJ, Mirkin CA, Smith JC, Mrksich M. 2002. Protein nanoarrays generated by dip‐pen nanolithography. Science 295(5560): 1702–1705. [DOI] [PubMed] [Google Scholar]
  28. Manoil C, Traxler B. 2000. Insertion of in‐frame sequence tags into proteins using transposons. Methods 20(1): 55–61. [DOI] [PubMed] [Google Scholar]
  29. Mrksich M, Whitesides GM. 1996. Using self‐assembled monolayers to understand the interactions of man‐made surfaces with proteins and cells. Annu Rev Biophys Biomol Struct 25: 55–78. [DOI] [PubMed] [Google Scholar]
  30. Neves‐Petersen MT, Snabe T, Klitgaard S, Duroux M, Petersen SB. 2006. Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces. Protein Sci 15(2): 343–351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Norde W. 1986. Adsorption of proteins from solution at the solid‐liquid interface. Adv Colloid Interface Sci 25(4): 267–340. [DOI] [PubMed] [Google Scholar]
  32. Park TJ, Lee SY, Lee SJ, Park JP, Yang KS, Lee KB, Ko S, Park JB, Kim T, Kim SK, Shin YB, Chung BH, Ku SJ, Kim DH, Choi IS. 2006. Protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner. Anal Chem 78(20): 7197–7205. [DOI] [PubMed] [Google Scholar]
  33. Peelle BR, Krauland EM, Wittrup KD, Belcher AM. 2005. Design criteria for engineering inorganic material‐specific peptides. Langmuir 21(15): 6929–6933. [DOI] [PubMed] [Google Scholar]
  34. Presnova G, Grigorenko V, Egorov A, Ruzgas T, Lindgren A, Gorton L, Borchers T. 2000. Direct heterogeneous electron transfer of recombinant horseradish peroxidases on gold. Faraday Discuss 116(116): 281–289. [DOI] [PubMed] [Google Scholar]
  35. Prime KL, Whitesides GM. 1993. Adsorption of proteins onto surfaces containing end‐attached oligo(ethylene oxide)—A model system using self‐assembled monolayers. J Am Chem Soc 115(23): 10714–10721. [Google Scholar]
  36. Revzin A, Russell RJ, Yadavalli VK, Koh WG, Deister C, Hile DD, Mellott MB, Pishko MV. 2001. Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography. Langmuir 17(18): 5440–5447. [DOI] [PubMed] [Google Scholar]
  37. Rich A, Crick FHC. 1955. Structure of collagen. Nature 176(4489): 915–916. [DOI] [PubMed] [Google Scholar]
  38. Rusmini F, Zhong ZY, Feijen J. 2007. Protein immobilization strategies for protein biochips. Biomacromolecules 8(6): 1775–1789. [DOI] [PubMed] [Google Scholar]
  39. Sanchez C, Arribart H, Guille MMG. 2005. Biomimetism and bioinspiration as tools for the design of innovative materials and systems. Nat Mater 4(4): 277–288. [DOI] [PubMed] [Google Scholar]
  40. Sano KI, Sasaki H, Shiba K. 2005. Specificity and biomineralization activities of Ti‐binding peptide‐1 (TBP‐1). Langmuir 21(7): 3090–3095. [DOI] [PubMed] [Google Scholar]
  41. Sarikaya M, Tamerler C, Jen AKY, Schulten K, Baneyx F. 2003. Molecular biomimetics: Nanotechnology through biology. Nat Mater 2(9): 577–585. [DOI] [PubMed] [Google Scholar]
  42. Schessler HM, Karpovich DS, Blanchard GJ. 1996. Quantitating the balance between enthalpic and entropic forces in alkanethiol/gold monolayer self assembly. J Am Chem Soc 118(40): 9645–9651. [Google Scholar]
  43. Seker UOS, Wilson B, Dincer S, Kim IW, Oren EE, Evans JS, Tamerler C, Sarikaya M. 2007. Adsorption behavior of linear and cyclic genetically engineered platinum binding peptides. Langmuir 23(15): 7895–7900. [DOI] [PubMed] [Google Scholar]
  44. Shao WH, Zhang XE, Liu H, Zhang ZP. 2000. Anchor‐chain molecular system for orientation control in enzyme immobilization. Bioconjug Chem 11(6): 822–826. [DOI] [PubMed] [Google Scholar]
  45. Sicheri F, Yang DSC. 1995. Ice‐binding structure and mechanism of an antifreeze protein from winter flounder. Nature 375(6530): 427–431. [DOI] [PubMed] [Google Scholar]
  46. Slocik JM, Wright DW. 2003. Biomimetic mineralization of noble metal nanoclusters. Biomacromolecules 4(5): 1135–1141. [DOI] [PubMed] [Google Scholar]
  47. Tamerler C, Duman M, Oren EE, Gungormus M, Xiong XR, Kacar T, Parviz BA, Sarikaya M. 2006a. Materials specificity and directed assembly of a gold‐binding peptide. Small 2(11): 1372–1378. [DOI] [PubMed] [Google Scholar]
  48. Tamerler C, Oren EE, Duman M, Venkatasubramanian E, Sarikaya M. 2006b. Adsorption kinetics of an engineered gold binding peptide by surface plasmon resonance spectroscopy and a quartz crystal microbalance. Langmuir 22(18): 7712–7718. [DOI] [PubMed] [Google Scholar]
  49. Tominaga J, Kamiya N, Doi S, Ichinose H, Maruyama T, Goto M. 2005. Design of a specific peptide tag that affords covalent and site‐specific enzyme immobilization catalyzed by microbial transglutaminase. Biomacromolecules 6(4): 2299–2304. [DOI] [PubMed] [Google Scholar]
  50. Tomizaki KY, Usui K, Mihara H. 2005. Protein‐detecting microarrays: Current accomplishments and requirements. Chembiochem 6(5): 783–799. [DOI] [PubMed] [Google Scholar]
  51. Watzke A, Kohn M, Gutierrez‐Rodriguez M, Wacker R, Schroder H, Breinbauer R, Kuhlmann J, Alexandrov K, Niemeyer CM, Goody RS, Waldmann H. 2006. Site‐selective protein immobilization by Staudinger ligation. Angew Chem Int Ed 45(9): 1408–1412. [DOI] [PubMed] [Google Scholar]
  52. Whaley SR, English DS, Hu EL, Barbara PF, Belcher AM. 2000. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 405(6787): 665–668. [DOI] [PubMed] [Google Scholar]
  53. Willey TM, Vance AL, van Buuren T, Bostedt C, Terminello LJ, Fadley CS. 2005. Rapid degradation of alkanethiol‐based self‐assembled monolayers on gold in ambient laboratory conditions. Surf Sci 576(1–3): 188–196. [Google Scholar]
  54. Woodbury RG, Wendin C, Clendenning J, Melendez J, Elkind J, Bartholomew D, Brown S, Furlong CE. 1998. Construction of biosensors using a gold‐binding polypeptide and a miniature integrated surface plasmon resonance sensor. Biosens Bioelectron 13(10): 1117–1126. [DOI] [PubMed] [Google Scholar]
  55. Xia YN, Whitesides GM. 1998. Soft lithography. Annu Rev Mater Sci 28: 153–184. [Google Scholar]
  56. Zhang JK, Cass AEG. 2001. A study of his‐tagged alkaline phosphatase immobilization on a nanoporous nickel‐titanium dioxide film. Anal Biochem 292(2): 307–310. [DOI] [PubMed] [Google Scholar]
  57. Zhen GL, Falconnet D, Kuennemann E, Voros J, Spencer ND, Textor M, Zurcher S. 2006. Nitrilotriacetic acid functionalized graft copolymers: A polymeric interface for selective and reversible binding of histidine‐tagged proteins. Adv Funct Mater 16(2): 243–251. [Google Scholar]
  58. Zhou HX, Dill KA. 2001. Stabilization of proteins in confined spaces. Biochemistry 40(38): 11289–11293. [DOI] [PubMed] [Google Scholar]
  59. Zin MT, Ma H, Sarikaya M, Jen AKY. 2005. Assembly of gold nanoparticles using genetically engineered polypeptides. Small 1(7): 698–702. [DOI] [PubMed] [Google Scholar]

Articles from Biotechnology and Bioengineering are provided here courtesy of Wiley

RESOURCES