Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli

BMC Biotechnology

Volumn 13, Issue , 2013, Pages

  Lobo, Marina Duarte Pinto ^a   Silva, Fredy Davi Albuquerque ^a   Landim, Patrícia Gadelha de Castro ^a   da Cruz, Paloma Ribeiro ^a   de Brito, Thaís Lima ^a   de Medeiros, Suelen Carneiro ^a   Oliveira, José Tadeu Abreu ^a   Vasconcelos, Ilka Maria ^a   Pereira, Humberto D'Muniz ^b   Grangeiro, Thalles Barbosa ^a  

^a FEDERAL UNIVERSITY OF CEARÁ   (Brazil)

^b UNIVERSITY OF SÃO PAULO   (Brazil)

Author keywords

Chitin binding domain; Chitinase; Heterologous; Secretion; Signal peptide

Indexed keywords

CHITIN-BINDING DOMAINS; CHITINASES; HETEROLOGOUS; SECRETION; SIGNAL PEPTIDE;

AFFINITY CHROMATOGRAPHY; BIOCONTROL; CLONING; ELECTROPHORESIS; ENCODING (SYMBOLS); ENZYMES; ESCHERICHIA COLI; GENE ENCODING; HYDROLYSIS; PHYSIOLOGY; PURIFICATION; RECOMBINANT PROTEINS;

CHITIN;

4 NITROPHENYL N,N' DIACETYLCHITOBIOSE; 4 NITROPHENYL N,N',N'' TRIACETYLCHITOTRIOSE; BINDING PROTEIN; CHITIN BINDING PROTEIN; CHITINASE; CHITOBIOSE; GLYCOSIDASE; RECOMBINANT PROTEIN; SIGNAL PEPTIDE; TRIOSE; UNCLASSIFIED DRUG;

AFFINITY CHROMATOGRAPHY; AMINO TERMINAL SEQUENCE; ANIMAL CELL; ARTICLE; BACTERIAL CELL; BACTERIAL STRAIN; CARBOXY TERMINAL SEQUENCE; CATALYSIS; CELL FREE SYSTEM; CHROMOBACTERIUM VIOLACEUM; CONTROLLED STUDY; DENATURING GRADIENT GEL ELECTROPHORESIS; ENZYME RELEASE; ENZYME SYNTHESIS; ESCHERICHIA COLI; HEATING; HYDROLYSIS; MOLECULAR WEIGHT; NONHUMAN; NUCLEOTIDE SEQUENCE; OPEN READING FRAME; PH; POLYMERASE CHAIN REACTION; PROTEIN DOMAIN; PROTEIN EXPRESSION; PROTEIN PURIFICATION; RECOMBINANT PLASMID; SUPERNATANT;

AMINO ACID SEQUENCE; CHITINASE; CHROMATOGRAPHY, AFFINITY; CHROMOBACTERIUM; CLONING, MOLECULAR; ESCHERICHIA COLI; GENETIC VECTORS; MOLECULAR SEQUENCE DATA; OPEN READING FRAMES; RECOMBINANT PROTEINS; SEQUENCE ALIGNMENT; SUBSTRATE SPECIFICITY;

ARTHROPODA; CHROMOBACTERIUM VIOLACEUM; ESCHERICHIA COLI; FUNGI; MOLLUSCA; PROTEOBACTERIA;

EID: 84878374033     PISSN: None     EISSN: 14726750     Source Type: Journal    
DOI: 10.1186/1472-6750-13-46     Document Type: Article

References (83)

1. Muzzarelli RAA. Native, industrial, and fossil chitins. Chitin and chitinases 1999, Basel: Birkhäuser, Jollès P, Muzzarelli RAA.
2. Hegedus D, Erlandson M, Gillott C, Toprak U. New insights into peritrophic matrix synthesis, architecture, and function. Annu Rev Entomol 2009, 54:285-302. 10.1146/annurev.ento.54.110807.090559, 19067633.
3. Ubhayasekera W. Structure and function of chitinases from glycoside hydrolase family 19. Polym Int 2011, 60:890-896.
4. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1991, 280(Pt 2):309-316. 1130547, 1747104.
5. Funkhouser JD, Aronson NN. Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family. BMC Evol Biol 2007, 7:96. 10.1186/1471-2148-7-96, 1945033, 17594485.
6. Udaya Prakash NA, Jayanthi M, Sabarinathan R, Kangueane P, Mathew L, Sekar K. Evolution, homology conservation, and identification of unique sequence signatures in GH19 family chitinases. J Mol Evol 2010, 70:466-478. 10.1007/s00239-010-9345-z, 20480157.
7. Iseli B, Armand S, Boller T, Neuhaus JM, Henrissat B. Plant chitinases use two different hydrolytic mechanisms. FEBS Lett 1996, 382:186-188. 10.1016/0014-5793(96)00174-3, 8612749.
8. Tews I, Terwisscha Van Scheltinga AC, Perrakis A, Wilson KS, Dijkstra BW. Substrate-assisted catalysis unifies two families of chitinolytic enzymes. J Am Chem Soc 1997, 119:7954-7959.
9. Synstad B, Gåseidnes S, Van Aalten DMF, Vriend G, Nielsen JE, Eijsink VGH. Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase. Eur J Biochem 2004, 271:253-262. 10.1046/j.1432-1033.2003.03923.x, 14717693.
10. Perrakis A, Tews I, Dauter Z, Oppenheim AB, Chet I, Wilson KS, Vorgias CE. Crystal structure of a bacterial chitinase at 2.3 Å resolution. Structure 1994, 2:1169-1180. 10.1016/S0969-2126(94)00119-7, 7704527.
11. Seidl V. Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions. Fungal Biol Rev 2008, 22:36-42.
12. Van Loon LC, Rep M, Pieterse CMJ. Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 2006, 44:135-162. 10.1146/annurev.phyto.44.070505.143425, 16602946.
13. Aam BB, Heggset EB, Norberg AL, Sørlie M, Vårum KM, Eijsink VGH. Production of chitooligosaccharides and their potential applications in medicine. Mar Drugs 2010, 8:1482-1517. 10.3390/md8051482, 2885077, 20559485.
14. Chen J-K, Shen C-R, Liu C-L. N-acetylglucosamine: production and applications. Mar Drugs 2010, 8:2493-2516. 10.3390/md8092493, 2953398, 20948902.
15. Koburger JA, May SO. Isolation of Chromobacterium spp. from foods, soil, and water. Appl Environ Microbiol 1982, 44:1463-1465. 242212, 7159087.
16. Vasconcelos ATR, Almeida de DF, Hungria M, Guimarães CT, Antônio RV, Almeida FC, Almeida LGP, Almeida R, Avles-Gomes JA, Andrade EM, Araripe J, Araújo MFF, Astolfi-Filho S, Azevedo V, Baptista AJ, Bataus LAM, Batista Da JS, Beló A, Van den Berg C, Bogo M, Bonatto S, Bordignon J, Brigido MM, Brito CA, Brocchi M, Burity HA, Camargo AA, Cardoso D das D de P, Carneiro NP, Carraro DM, et al. The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc Natl Acad Sci U S A 2003, 100:11660-11665. 208814, 14500782.
17. Grangeiro TB, Jorge DM de M, Bezerra WM, Vasconcelos ATR, Simpson AJG. Transport genes of Chromobacterium violaceum: an overview. Genet Mol Res 2004, 3:117-133.
18. Ikegami T, Okada T, Hashimoto M, Seino S, Watanabe T, Shirakawa M. Solution structure of the chitin-binding domain of Bacillus circulans WL-12 chitinase A1. J Biol Chem 2000, 275:13654-13661. 10.1074/jbc.275.18.13654, 10788483.
19. Hashimoto M, Honda Y, Nikaidou N, Fukamizo T, Watanabe T. Site-directed mutagenesis of Asp280 suggests substrate-assisted catalysis of chitinase A1 from Bacillus circulans WL-12. J Biosci Bioeng 2000, 89:100-102. 10.1016/S1389-1723(00)90031-8, 16232709.
20. Ferrandon S, Sterzenbach T, Mersha FB, Xu M-Q. A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag. Biochim Biophys Acta 2003, 1621:31-40. 10.1016/S0304-4165(03)00029-1, 12667608.
21. Hardt M, Laine RA. Mutation of active site residues in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: use of a green fluorescent protein binding assay. Arch Biochem Biophys 2004, 426:286-297. 10.1016/j.abb.2004.03.017, 15158679.
22. Watanabe T, Ito Y, Yamada T, Hashimoto M, Sekine S, Tanaka H. The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J Bacteriol 1994, 176:4465-4472. 196264, 8045877.
23. Hashimoto M, Ikegami T, Seino S, Ohuchi N, Fukada H, Sugiyama J, Shirakawa M, Watanabe T. Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J Bacteriol 2000, 182:3045-3054. 10.1128/JB.182.11.3045-3054.2000, 94488, 10809681.
24. Gilkes NR, Henrissat B, Kilburn DG, Miller RC, Warren RA. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev 1991, 55:303-315. 372816, 1886523.
25. Poon DKY, Withers SG, McIntosh LP. Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi Xylanase Cex through NMR spectroscopic analysis. J Biol Chem 2007, 282:2091-2100.
26. Vaaje-Kolstad G, Houston DR, Rao FV, Peter MG, Synstad B, Van Aalten DMF, Eijsink VGH. Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin. Biochim Biophys Acta 2004, 1696:103-111. 10.1016/j.bbapap.2003.09.014, 14726210.
27. Van Aalten DM, Synstad B, Brurberg MB, Hough E, Riise BW, Eijsink VG, Wierenga RK. Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-Å resolution. Proc Natl Acad Sci U S A 2000, 97:5842-5847. 10.1073/pnas.97.11.5842, 18521, 10823940.
28. Brameld KA, Goddard WA. The role of enzyme distortion in the single displacement mechanism of family 19 chitinases. Proc Natl Acad Sci U S A 1998, 95:4276-4281. 10.1073/pnas.95.8.4276, 22479, 9539727.
29. Brameld KA, Shrader WD, Imperiali B, Goddard WA. Substrate assistance in the mechanism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors. J Mol Biol 1998, 280:913-923. 10.1006/jmbi.1998.1890, 9671559.
30. Van Aalten DM, Komander D, Synstad B, Gåseidnes S, Peter MG, Eijsink VG. Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci U S A 2001, 98:8979-8984. 10.1073/pnas.151103798, 55359, 11481469.
31. Hsieh Y-C, Wu Y-J, Chiang T-Y, Kuo C-Y, Shrestha KL, Chao C-F, Huang Y-C, Chuankhayan P, Wu W-G, Li Y-K, Chen C-J. Crystal structures of Bacillus cereus NCTU2 chitinase complexes with chitooligomers reveal novel substrate binding for catalysis: a chitinase without chitin binding and insertion domains. J Biol Chem 2010, 285:31603-31615. 10.1074/jbc.M110.149310, 2951234, 20685646.
32. Li H, Greene LH. Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding. PLoS One 2010, 5:e8654. 10.1371/journal.pone.0008654, 2805709, 20084296.
33. Liu T, Yan J, Yang Q. Comparative biochemistry of GH3, GH20 and GH84 β-N-acetyl-Dhexosaminidases and recent progress in selective inhibitor discovery. Curr Drug Targets 2012, 13:512-525. 10.2174/138945012799499730, 22280348.
34. Keyhani NO, Roseman S. Physiological aspects of chitin catabolism in marine bacteria. Biochim Biophys Acta 1999, 1473:108-122. 10.1016/S0304-4165(99)00172-5, 10580132.
35. Gooday G. The ecology of chitin degradation. Adv Microb Ecol 1990, 11:387-430.
36. Streichsbier F. Utilization of chitin as sole carbon and nitrogen source by Chromobacterium violaceum. FEMS Microbiol Lett 1983, 19:129-132.
37. Chernin LS, Winson MK, Thompson JM, Haran S, Bycroft BW, Chet I, Williams P, Stewart GS. Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing. J Bacteriol 1998, 180:4435-4441. 107452, 9721280.
38. Sitrit Y, Vorgias CE, Chet I, Oppenheim AB. Cloning and primary structure of the chiA gene from Aeromonas caviae. J Bacteriol 1995, 177:4187-4189. 177160, 7608101.
39. Chen JP, Nagayama F, Chang MC. Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli. Appl Environ Microbiol 1991, 57:2426-2428. 183590, 1768115.
40. Tsujibo H, Orikoshi H, Tanno H, Fujimoto K, Miyamoto K, Imada C, Okami Y, Inamori Y. Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7. J Bacteriol 1993, 175:176-181. 196111, 8416892.
41. Wang SY, Wu SJ, Thottappilly G, Locy RD, Singh NK. Molecular cloning and structural analysis of the gene encoding Bacillus cereus exochitinase Chi36. J Biosci Bioeng 2001, 92:59-66.
42. Watanabe T, Suzuki K, Oyanagi W, Ohnishi K, Tanaka H. Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. J Biol Chem 1990, 265:15659-15665.
43. Chernin LS, De la Fuente L, Sobolev V, Haran S, Vorgias CE, Oppenheim AB, Chet I. Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans. Appl Environ Microbiol 1997, 63:834-839. 168378, 9055404.
44. Gleave AP, Taylor RK, Morris BA, Greenwood DR. Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum. FEMS Microbiol Lett 1995, 131:279-288. 10.1111/j.1574-6968.1995.tb07788.x, 7557339.
45. Jones JD, Grady KL, Suslow TV, Bedbrook JR. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J 1986, 5:467-473. 1166787, 16453672.
46. Robbins PW, Albright C, Benfield B. Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli. J Biol Chem 1988, 263:443-447.
47. Kadokura K, Sakamoto Y, Saito K, Ikegami T, Hirano T, Hakamata W, Oku T, Nishio T. Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium. Biosci Biotechnol Biochem 2007, 71:2848-2851. 10.1271/bbb.70389, 17986788.
48. Cronin D, MoenneLoccoz Y, Dunne C, OGara F. Inhibition of egg hatch of the potato cyst nematode Globodera rostochiensis by chitinase-producing bacteria. Eur J Plant Pathol 1997, 103:433-440.
49. Park SK, Lee M-C, Harman GE. The biocontrol activity of chromobacterium sp. strain C-61 against rhizoctonia solani depends on the productive ability of chitinase. Plant Pathol J 2005, 21:275-282.
50. Wynn-Williams DD. Distribution and characteristics of Chromobacterium in the maritime and sub-antarctic. Polar Biol 1983, 2:101-108.
51. Tata SJ, Beintema JJ, Balabaskaran S. The lysozyme of Hevea brasiliensis latex - isolation, purification, enzyme kinetics and a partial amino acid sequence. J Rubber Res Inst Malays 1983, 31:35-48.
52. Hoell IA, Klemsdal SS, Vaaje-Kolstad G, Horn SJ, Eijsink VGH. Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1. Biochim Biophys Acta 2005, 1748:180-190. 10.1016/j.bbapap.2005.01.002, 15769595.
53. Perkins SJ, Johnson LN, Machin PA, Phillips DC. Crystal structures of hen egg-white lysozyme complexes with gadolinium(III) and gadolinium(III)-N-acetyl-D-glucosamine. Biochem J 1979, 181:21-36. 1161121, 486153.
54. De Vrije T, Batenburg AM, Jordi W, De Kruijff B. Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation. Eur J Biochem 1989, 180:385-392. 10.1111/j.1432-1033.1989.tb14660.x, 2466667.
55. Phoenix DA, Kusters R, Hikita C, Mizushima S, De Kruijff B. OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane. J Biol Chem 1993, 268:17069-17073.
56. Puziss JW, Fikes JD, Bassford PJJ. Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide. J Bacteriol 1989, 171:2303-2311. 209902, 2651397.
57. Akita M, Sasaki S, Matsuyama S, Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem 1990, 265:8164-8169.
58. Chou Y-T, Gierasch LM. The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA. J Biol Chem 2005, 280:32753-32760. 10.1074/jbc.M507532200, 16046390.
59. Gelis I, Bonvin AMJJ, Keramisanou D, Koukaki M, Gouridis G, Karamanou S, Economou A, Kalodimos CG. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 2007, 131:756-769. 10.1016/j.cell.2007.09.039, 2170882, 18022369.
60. Musial-Siwek M, Rusch SL, Kendall DA. Selective photoaffinity labeling identifies the signal peptide binding domain on SecA. J Mol Biol 2007, 365:637-648. 10.1016/j.jmb.2006.10.027, 1851904, 17084862.
61. Von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 1983, 133:17-21. 10.1111/j.1432-1033.1983.tb07424.x, 6852022.
62. Kajava AV, Zolov SN, Kalinin AE, Nesmeyanova MA. The net charge of the first 18 residues of the mature sequence affects protein translocation across the cytoplasmic membrane of gram-negative bacteria. J Bacteriol 2000, 182:2163-2169. 10.1128/JB.182.8.2163-2169.2000, 111264, 10735858.
63. Mergulhão FJM, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli. Biotechnol Adv 2005, 23:177-202. 10.1016/j.biotechadv.2004.11.003, 15763404.
64. Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 2004, 64:625-635. 10.1007/s00253-004-1559-9, 14966662.
65. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402. 10.1093/nar/25.17.3389, 146917, 9254694.
66. Bendtsen JD, Nielsen H, Von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004, 340:783-795. 10.1016/j.jmb.2004.05.028, 15223320.
67. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook 2005, 571-607. New York: Humana Press, Walker JM.
68. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Lu S, Marchler GH, Song JS, Thanki N, Yamashita RA, Zhang D, Bryant SH. CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res 2013, 41:D348-D352. 10.1093/nar/gks1243, 3531192, 23197659.
69. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. 10.1093/nar/22.22.4673, 308517, 7984417.
70. Holm L, Rosenström P. Dali server: conservation mapping in 3D. Nucleic Acids Res 2010, 38:W545-W549. 10.1093/nar/gkq366, 2896194, 20457744.
71. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 2009, 37:D233-D238. 10.1093/nar/gkn663, 2686590, 18838391.
72. Warner SAJ. Genomic DNA Isolation and Lambda Library Construction. Plant Gene Isolation: principle and practice 1996, 51-73. West Sussex: John Wiley & Sons, Foster GD, Twell D.
73. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual 1989, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2.
74. Koshland D, Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell 1980, 20:749-760. 10.1016/0092-8674(80)90321-9, 6448092.
75. Boller T. Biochemical analysis of chitinase and β-1,3-glucanases. Molecular Plant Pathology: A Practical Approach 1992, 23-30. New York: Oxford University Press, Gurr S, McPherson M, Bowles D.
76. Molano J, Durán A, Cabib E. A rapid and sensitive assay for chitinase using tritiated chitin. Anal Biochem 1977, 83:648-656. 10.1016/0003-2697(77)90069-0, 603047.
77. Boller T, Gehri A, Mauch F, Vogeli U. Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. Planta 1983, 157:22-31.
78. Reissig JL, Storminger JL, Leloir LF. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 1955, 217:959-966.
79. Frändberg E, Schnürer J. Evaluation of a chromogenic chito-oligosaccharide analogue, p-nitrophenyl-beta-D-N, N'-diacetylchitobiose, for the measurement of the chitinolytic activity of bacteria. J Appl Bacteriol 1994, 76:259-263. 10.1111/j.1365-2672.1994.tb01625.x, 8157546.
80. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254. 10.1016/0003-2697(76)90527-3, 942051.
81. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685. 10.1038/227680a0, 5432063.
82. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979, 76:4350-4354. 10.1073/pnas.76.9.4350, 411572, 388439.
83. Edman P. Method for determination of the amino acid sequence in peptides. Acta Chem Scand 1950, 4:283-293.