Biotecnología de Proteínas Recombinantes para la Aplicación en Acuacultura
Resumen
Las Ciencias del Mar y la acuacultura no han quedado al margen de la aplicación de los nuevos avances
alcanzados en la Biotecnología Molecular y ésta última ha jugado un papel muy importante en el desarrollo de
la Biotecnología de proteínas ya que gracias a la manipulación de genes ha sido posible producir grandes
cantidades de proteínas, muchas de éstas presentes en concentraciones muy bajas en su ambiente natural.
En este trabajo se describen los enfoques que la acuacultura ha dado a la producción de proteínas
recombinantes. También se analizan los criterios requeridos para la selección de un sistema de expresión para
producir estas proteínas y se comparan los hospederos unicelulares más empleados para estos fines.
Por último se analizan las propiedades de tripsinas y tripsinógenos de especies acuáticas en especial del
Camarón Blanco del Pacífico (Litopenaeus vannamei) y los resultados obtenidos con el empleo de Pichia
pastoris para la producción la forma recombinante de esta proteína de gran interés en la acuacultura.
Descargas
Citas
Aoki H., Ahsan M.N., Watabe S. (2003) Heterologous expression in Pichia pastoris and single-step
purification of a cysteine proteinase from northern shrimp. Protein Expression and Purification 31,
-221.
Bi X.Z., Chew F.T. (2004) Molecular, proteomic and immunological characterization of isoforms of arginine
kinase, a cross-reactive invertebrate pan-allergen, from the house dust mite, Dermatophagoides
farinae. Journal of Allergy and Clinical Immunology 113, S226.
Buckholz R.G., Gleeson M.A. (1991) Yeast systems for the commercial production of heterologous proteins.
Biotechnology 9, 1067-1072.
Cereghino G.P.L., Cregg J.M. (1999) Applications of yeast in biotechnology: Protein production and genetic
analysis. Current Opinion Biotechnology 10, 422–427.
Cereghino G.P.L., Cregg J.M. (2000) Heterologous protein expression in the methylotrophic yeast Pichia
pastoris. FEMS Microbiology Reviews 24, 45-66.
Chan Y.H., Cheng C.H.K., Chan K.M. (2003) Recombinant goldfish growth hormones (gfGH-I and -II)
expressed in Escherichia coli have similar biological activities. Comparative Biochemistry and
Physiology - Part A: Molecular & Integrative Physiology 135, 613-624.
Chen L-L., Leu J-H., Huang C-J., Chou C-M., Chen S-M., Wang C-H., Lo C-F., Kou G-H. (2002)
Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and
characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells.
Virology 293, 44-53.
Cohen T., Gertler A. (1981) Pancreatic proteolytic enzymes from carp Cyprinus carpio I. Purification and
physical properties of trypsin, chymosin, elastase and carboxypeptidase B. Comparative
Biochemistry and Physiology 69B, 647–653.
Cregg J.M., Vedvick T.S., Rascke W.C. (1993) Recent advances in the expression of foreign genes in Pichia
pastoris. Biotechnology 11, 905-910.
De-Vecchi S.D., Coppes Z. (1996) Marine fish digestive proteases —relevance to food industry and southwest
Atlantic region—a review. Journal of Food Biochemistry 20, 193–214.
Eckart M.R., Bussineau C.M. (1996) Quality and authenticity of heterologous proteins synthesized in yeast.
Current Opinion Biotechnology 7, 525-530.
Escamilla-Treviño L.L. (2002) Producción de tripsina del camarón blanco del Pacífico (Penaeus vannamei)
en Pichia pastoris. Tesis de Doctorado en Ciencias con especialidad en Biotecnología de la Facultad
de Ciencias Biológicas, U.A.N.L., Monterrey, N.L. (México).
Escamilla-Treviño L.L., Viader-Salvadó J.M., Guerrero-Olazarán M. (1999) Producción de proteínas
recombinantes en Pichia pastoris. CIENCIA UANL 2, 27-33.
Galgani F.G., Benyamin Y., Van Wormhoudt A. (1985) Purification, properties and immunoassay of trypsin
from the shrimp Penaeus japonicus. Comparative Biochemistry and Physiology 81B, 447-452.
Gallegos-López J.A. (2004) Síntesis y clonación molecular del DNAc de α-glucosidasa de camarón
Litopenaeus vannamei. Tesis de Licenciatura de la carrera de Químico Bacteriólogo Parasitólogo de
la Facultad de Ciencias Biológicas, U.A.N.L., San Nicolás de los Garza, N.L. (México).
Gates B.J., Travis J. (1969) Isolation and comparative properties of shrimp trypsin (Penaeus setiferus).
Biochemistry 8, 4483-4489.
Gellisen G., Hollenberg C. (1997) Application of yeasts in gene expression studies: a comparison of
Saccharomyces cerevisiae, Hansenula polymorpha, and Kluyveromyces lactis — a review. Gene
, 87-97.
Gu P.-L., Yu K.L., Chan S-M. (2000) Molecular characterization of an additional shrimp hyperglycemic
hormone: cDNA cloning, gene organization, expression and biological assay of recombinant
proteins. FEBS Letters 472, 122-128.
Guerrero-Olazarán M., Viader-Salvadó J.M. (2003) Pichia pastoris como hospedero para la producción de
proteínas recombinantes. In: Procesos Biotecnológicos. (ed. by Galán-Wong L.J., Elías-Santos M.,
Tamez-Guerra P., Quintero-Ramírez R., Quintero-Zapata I.), pp. 136–156. Universidad Autónoma
de Nuevo León. San Nicolás de los Garza.
Guyonnet V., Thuscik F., Long P.L., Polanowski L.A., Travis J. (1999) Purification and partial
characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin and elastase from the
chicken. Journal of Chromatography A 852, 217–225.
Haard N.F. (1992) A review of proteolytic enzymes from marine organisms and their application in the food
industry. Journal of Aquatic Food Product Technology 1, 17–35.
Haard N.F. (1994). Protein hydrolysis in seafoods. In: Seafoods chemistry, processing technology and quality
(ed. by Shahidi F., Botta J.R.), pp. 11–33. Blackie Academic and Professional, Glasgow, UK.
Haard N.F. (1998). Speciality enzymes from marine organisms. Food Technology 53, 64–67.
Haard N.F., Simpson B.K. (1994). Proteases from aquatic organisms and their uses in the seafood industry.
In: Fisheries processing: biotechnological applications (ed. by Martin A.M.), pp. 132–154.
Chapman and Hall, London, UK.
Hanquier J., Sorlet Y., Desplancq D., Baroche L., Ebtinger M., Lefèvre J.F., Pattus F.,. Hershberger C.L,
Vertès A.A. (2003) A single mutation in the activation site of bovine trypsinogen enhances its
accumulation in the fermentation broth of the yeast Pichia pastoris. Applied and Environmental
Microbiology 69, 1108–1113.
Hanquier J.M., Hbrshberger C.L., Desplanca D., Lardson J.L., Rosteck P.R. (2000) Production of soluble
recombinant trypsinogen analogs. WO 00/17332, PCT/US99/21047.
Higaki J.N., Evin L.B., Craik C.S. (1989). Introduction of a cysteine protease active site into trypsin.
Biochemistry 28, 9256-9263.
Higgins D.R., Cregg J.M. (1998) Introduction to Pichia pastoris. In: Pichia Protocols. Methods in Molecular
Biology 103 (ed. by Higgins D.R., Cregg J.M.), Humana Press, Totowa, NJ.
Honjo I., Kimura S., Nonaka M. (1990) Purification and characterization of trypsin-like enzyme from shrimp
Penaeus indicus. Nippon Suisan Gakkaishi 56(10), 1627-1634.
Huang C-C., Sritunyalucksana K., Söderhäll K., Song Y-L. (2004) Molecular cloning and characterization of
tiger shrimp (Penaeus monodon) transglutaminase Developmental & Comparative Immunology 28,
-294.
Jiang S.T., Moody M., Chen H.C. (1991) Purification and characterization of proteases from digestive tract of
grass shrimp (Penaeus monodon). Journal of Food Science 56, 322–326.
Jónsdóttir G., Bjarnason J.B, Gudmundsdóttir Á. (2004) Recombinant cold-adapted trypsin I from Atlantic
cod—expression, purification, and identification. Protein Expression and Purification 33, 110-122.
Keil B. (1971) Trypsin. In: The Enzymes. 3rd ed. Vol. III. pp. 249-275.Academic Press. New York.
Kim D.K., Jang I.K., Seo H.C., Shin S.O., Yang S.Y., Kim J.W. (2004) Shrimp protected from WSSV disease
by treatment with egg yolk antibodies (IgY) against a truncated fusion protein derived from WSSV.
Aquaculture 237, 21-30.
Klein B., Moullac L.G., Sellos D., Wormhoudt A.V. (1996) Molecular Cloning and Sequencing of Trypsin
cDNAs from Penaeus vannamei (Crustacea, Decapoda): Use in Assessing Gene Expression during
the Moult Cycle. Journal. Biochemestry. Cell biology. 28(5), 551-563.
Le Moullac (1994) Adaptation des enzymes digestives à l’alimentation chez la crevette Penaeus vannamei
(Crustacea, Decapoda). Memoire pour l’optention du diplôme de l’Ecole Practique des Hautes
Etudes. Montpellier (France).
Le Moullac G.L., Klein B., Sellos D., van Wormhoudt A. (1996) Adaptation of trypsin, chymotrypsin and α-
amylase to casein level and protein source in Penaeus vannamei (Crustacea Decapoda). Journal of
Experimental Marine Biology and Ecology 208, 107–125.
Lee P.G., Lawrence A.L. (1982) A quantitative analysis of digestive enzymes in penaeid shrimp; influence of
diet, age and species. Physiologist 25, 241
Lin S-T., Chang Y-S., Wang H-C., Tzeng H-F., Chang Z-F., Lin J-Y., Wang C-H., Lo C-F., Kou G-H. (2002)
Ribonucleotide reductase of shrimp white spot syndrome virus (WSSV): expression and enzymatic
activity in a baculovirus/insect cell system and WSSV-infected shrimp. Virology 304, 282-290.
Lu P.J., Liu H.C., Tsai I.H. (1990) The midgut trypsins of shrimp (Penaeus monodon), biology and chemistry.
Hoppe-Seyler 371, 851–857.
Luo T., Zhang X., Shao Z., Xu X. (2003) PmAV, a novel gene involved in virus resistance of shrimp. Penaeus
monodon FEBS Letters 551, 53-57.
Macouzet M., Simpson B.K., Lee B.H (1999) Cloning of fish enzymes and other fish protein genes. Critical
Reviews in Biotechnology 19, 179-196.
Martínez A., Olsen R.L. Serra J.L. (1988) Purification and characterization of two trypsin-like enzymes from
the digestive tract of anchovy Engraulis encrasicholus. Comparative Biochemistry and Physiology A
Molecular and Integrative Physiol 91, 677–684.
McLean E, Devlin R.H., Byatt J.C., Clarke W.C, Donaldson E.M (1997) Impact of a controlled release
formulation of recombinant bovine growth hormone upon growth and seawater adaptation in coho
(Oncorhynchus kisutch) and chinook (Oncorhynchus tshawytscha) salmon. Aquaculture 156, 113-
Melamed P., Gong Z., Fletcher G., Hew C.L. (2002) The potential impact of modern biotechnology on fish
aquaculture. Aquaculture 204, 255-269.
Muller S., Sandal S., Kamp-Hansen P., Dalboge H. 1998 Comparison of expression systems in the yeasts
Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces
pombe and Yarrowia lipolytica. Cloning of two novel promoters from Y. lipolytica. Yeast 14, 1267-
Neurath H. (1984) Evolution of proteolytic enzymes. Science 224, 350–357.
Ohta S., Nishikawa A., Imamura K. (2003) Molecular cloning and expression of pyruvate kinase from
globefish (Fugu rubripes) skeletal muscle. Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology 135, 397-405.
Peterson B.C., Small B.C.,. Bosworth B.G. (2004) Effects of bovine growth hormone (Posilac®) on growth
performance, body composition, and IGFBPs in two strains of channel catfish. Aquaculture 232,
-663.
Romanos M.A., Scorer C.A., Clare J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Romanos R. (1995) Advances in the use of Pichia pastoris for high level gene expression. Current Opinion
Biotechnology 6, 527-533.
Sainz J.C., García-Carreño F., Hernández-Cortés P. (2004a) Penaeus vannamei isotrypsins: purification and
characterization. Comparative Biochemistry and Physiology Part B 138, 155–162.
Sainz J.C., García-Carreño F., Sierra-Beltrán A., Hernández-Cortés P. (2004b) Trypsin synthesis and storage
as zymogen in the midgut gland of the shrimp Litopenaeus vannamei. Journal of Crustacean Biology
, 266–273.
Shahidi F., Janak Kamil Y.V.A. (2001). Enzymes from fish and aquatic invertebrates and their application in
the food industry. Trends in Food Science & Technology. 12, 435-464.
Tsai I.H., Lu P., Chuang J.L. (1991) The midgut enzymes of shrimp Penaeus monodon, Penaeus japonicus
and Penaeus penicillatus. Biochemica Biophysica 1080, 59–67.
Udomkit A., Treerattrakool S., Panyim S. (2004) Crustacean hyperglycemic hormones of Penaeus monodon:
cloning, production of active recombinant hormones and their expression in various shrimp tissues.
Journal of Experimental Marine Biology and Ecology 298, 79-91.
Viader-Salvadó J.M., Fuentes-Garibay J.A., Castillo-Galván M., Castillo-Galván M., Galán-Wong L.,
Guerrero-Olazarán M. (2004) Producción de tripsinógeno del camarón Litopenaeus vannamei en
cepas recombinantes Pichia pastoris a nivel fermentador. BIOTEC’04. Universidad de Oviedo y
Sociedad Española de Biotecnología, Oviedo (España).
Viader-Salvadó J.M., Guerrero-Olazarán M. (2003) Monitoreo y control de bioprocesos con Pichia pastoris.
En: Procesos Biotecnológicos. (ed. by Galán-Wong L.J., Elías-Santos M., Tamez-Guerra P.,
Quintero-Ramírez R., Quintero-Zapata I.), pp. 232-237, Universidad Autónoma de Nuevo León, San
Nicolás de los Garza.
Villacis J.F., Lehrer N., Vogel L., Schicktanz S., Randow S., Reese G., El-Dahr J.M., Vieths S., Lehrer S.B.
(2004) Comparison of IgE reactivity of recombinant and natural pen a 1 by RAST and mediator
release. Journal of Allergy and Clinical Immunology, 113, S148 .
Vozza L.A., Wittwer L., Higgins D.R., Purcell T.J., Beergseid M., Collins-Racie L.A., LaVallie E.R.,
Hoeffler J.P. (1996). Production of a recombinant bovine enterokinase catalytic subunit in the
methylotrophic yeast Pichia pastoris. Bio/Technology 14, 77-81.
Walsh G., Headon D.R. (1995) Protein Biotechnology. pp. 1, 13-15. John Wiley and Sons.
Walsh K. (1970) Trypsinogens and trypsins of various species. Methods in Enzymology 19, 41-63.
Woldike H.F., Kjeldsen T.B. (1999) Process for producing trypsin (trypsinogen). United States Patent
945.328.
Yee L., Blanch, H.W. (1993). Recombinant trypsin production in high cell density fed-batch cultures in
Escherichia coli. Biotechnology and Bioengineering 41, 781-790.
Yodmuang S., Udomkit A., Treerattrakool S., Panyim S. (2004) Molecular and biological characterization of
molt-inhibiting hormone of Penaeus monodon. Journal of Experimental Marine Biology and
Ecology 312, 101-114.
