2012 - Method To Concentrate Protein

Analytical Biochemistry 426 (2012) 4–12
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Method to concentrate protein solutions based on dialysis–freezing–centrifugation:

Enzyme applications
J.J. Virgen-Ortíz a,b, V. Ibarra-Junquera a,b,⇑, J.A. Osuna-Castro c,⇑, P. Escalante-Minakata b,
N.A. Mancilla-Margalli d, J. de J. Ornelas-Paz e
a
Faculty of Chemical Science, University of Colima, Coquimatlán, Col 28400, Mexico
b
Environmental Bioengineering Laboratory, Faculty of Civil Engineering (FIC), University of Colima, Colima, Col 28400, Mexico
c
Faculty of Biological and Agricultural Sciences, University of Colima, Tecomán, Col 28100, Mexico
d
División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Tlajomulco, Jalisco 45640, Mexico
e
Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD), Unidad Cuauhtémoc, Chihuahua 31570, Mexico
a r t i c l e i n f o a b s t r a c t
Article history: One of the recurrent methodological problems in preparative biochemical work is the concentration of
Received 26 December 2011 dilute protein solutions, including culture supernatants resulting from biotechnological processes. A pro-
Received in revised form 22 March 2012 cedure was developed to concentrate enzymes by a novel cryoconcentration system. This approach
Accepted 23 March 2012
includes a new device that facilitates the sample freezing and the subsequent solute elution from the fro-
Available online 1 April 2012
zen matrix by centrifugation. The optimal centrifugation conditions for this cryoconcentration system
were obtained using whey protein solution as a model. The procedure was applied to concentrate dilute
Keywords:
solutions of commercial pectinase, measuring the endopolygalacturonase (EPG) activity of this enzyme in
Cryoconcentration
Enzyme activity
the concentrate by a method based on the on-line torque measurement, and of recombinant fructan:fruc-
Freeze centrifugation tan 1-fructosyltransferase (1-FFT) protein of Pichia pastoris from a culture in a bioreactor, as an expression
Pectinase system. The optimal centrifugation speed, time, and temperature were 6150 g, 20 min, and 4 °C, respec-
Milk whey tively. The concentration factors for the dilute protein solutions were 9.2-, 11.2-, and 17.1-fold for 1-FFT,
whey, and commercial pectinase, respectively. Recoveries ranged from 87% to 93%. The procedure
allowed concentrating proteins efficiently without affecting their enzymatic activity.
Ó 2012 Elsevier Inc. All rights reserved.
Nowadays, molecular biology and biotechnology, including scale are currently available, including lyophilization, chemical
large-scale proteomic technologies, are revolutionizing the precipitation, and ultrafiltration. However, certain technical prob-
production of therapeutic and many other products with high lems imposed by such methods have been reported. For example,
industrial value added, using diverse biochemical routes available, lyophilization of protein samples may require additional steps for
mainly in bacterial and yeast expression systems [1]. Consequently, the removal of salts and/or detergents [4]. Furthermore, protein
more and more research laboratories worldwide are investigating concentration by chemical precipitation is often incomplete and
and producing native or modified biomolecules such as proteins, ineffective with low recovery once the proteins have reached a con-
peptides, and antibodies for a wide range of medical, food, aca- centration below 1 mg/ml. In general, protein precipitation proto-
demic, and industrial applications. Protein concentration and puri- cols may be cumbersome due to incomplete precipitation or
fication methods are the key for many technologies that are difficult solubilization of the precipitated proteins [5]. Many pre-
transforming the way to learn about cellular processes. In cipitation methods, including precipitation with organic solvents
general, biomolecule purification involves a complex series of steps and isoelectric precipitation, can result in the denaturation of pro-
where targets are selectively separated through sequential teins or reduction of their bioactivity [6]. In addition, precipitation
processes [2,3]. During separation processes, the biomolecules methods are not selective for polypeptide molecules, and many
often need to be concentrated for the next purification step or times robust biomolecules such as carbohydrates, nucleic acids,
direct use. Many protein concentration methods at the laboratory and many structural proteins are also concentrated. The major dis-
advantage of ultrafiltration, however, is the formation of irrevers-
ible bind in some proteins. This may be especially detrimental
⇑ Corresponding authors. Address: Faculty of Chemical Science, University of
when only a few micrograms of proteins remain after a multistep
Colima, Coquimatlán, Col., Mexico. Fax: +52 312 3161167 (V. Ibarra-Junquera).
purification [5]. In general, for large-scale applications, there are
E-mail addresses: vij@ucol.mx (V. Ibarra-Junquera), josuna@ucol.mx
(J.A. Osuna-Castro). three main processes with common commercial application in
0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2012.03.019

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Analytical Biochemistry 426 (2012) 4–12

Contents lists available at SciVerse ScienceDirect

Method to concentrate protein solutions based on dialysis–freezing–centrifugation:

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