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Recombinant Proteins Unit

MEMBERS

  • Pablo Oppezzo, PhD (Head)
  • Agustín Correa, PhD (Principal technical assistant)
  • Claudia Ortega, PhD (Technical Assistant)
  • Cecilia Abreu, PhD (Technical Assistant)

MAIN EQUIPMENT

ÄKTAxpress

ÄKTAxpress for protein purification gives you the highest possible purity needed for structural and functional studies. Optimized protocols with a choice of up to four purification steps minimize the need for chromatography expertise. Tag removal and maintenance procedures can be integrated into the purification protocols, eliminating manual interference during a run. Purification schemes for double affinity-tagged proteins are also supported. A four-step protocol may consist of affinity chromatography (AC), desalting (DS), ion exchange chromatography (IEX) and gel filtration (GF).

ÄKTA Pure

ÄKTA pure is a flexible and intuitive chromatography system for fast purification of proteins, peptides, and nucleic acids from microgram levels to tens of grams of target product. Is a reliable system where hardware and UNICORN™ system control software are designed to work together with columns and media to meet any purification challenge.

ÄKTA Purifier 

ÄKTA™purifier systems are designed for fast, high-resolution separation and characterization of proteins at laboratory scale. The systems perform all chromatographic techniques and scout for optimal binding and elution conditions, pH, gradient shapes, and flow rates. This system can produce 25 MPa and flow rates up to 10 ml/min, become ideal for laboratory purification and high-resolution analysis.

Robot Tecan Genesis 200 

The Tecan Genesis 200 is a versatile robot for automating pipetting tasks. It is equipped with two arms, the LiHa (liquid handler) and the RoMa (robot manipulator). The LiHa is a conductivity-sensing 8-channel pipetting arm capable of dispensing volumes of 5-1000ul. The RoMa is a manipulating arm capable of picking up and moving objects on the workstation.

Benchtop Bioreactor BIOSTAT® B plus (Prokaryotic culture)

The BIOSTAT® B fermenter bioreactor has been specially designed to cover the wide variety of requirements in biotechnological and biopharmaceutical research and development.
The wide range of configurations is available to choose from for animal, plant and insect cell cultivation as well as for microbial fermentation. Volumes range from 1L to 2L to 5L.
Typical areas of application include the following:

  • Process development for the manufacture of vaccines, recombinant proteins and monoclonal antibodies
  • Process strategy development using a batch, fed-batch, continuous or perfusion mode
  • Scale-up and scale-down tests for commercial-scale manufacture
  • Small-scale (pre-) production
  • High cell-density fermentation
  • Adherent cell cultures on microcarriers
  • Low-shear-stress cell cultivation of sensitive organisms
  • Cultivation of filamentous organisms
  • Dual usage both for cell cultures and microbial applications, such as in academic research.

CelliGen 310 Bioreactor (Eukaryotic culture) 

CelliGen® 310 is a benchtop, autoclavable bioreactor with advanced controller and touchscreen interface capable of operating up to four reactors simultaneously.
The CelliGen 310 is an advanced benchtop cell culture bioreactor ideal for research through production. This powerful system can regulate up to 32 parameters each, in one to four vessels. Over 120 parameters total. So, you can integrate and control your own analyzers, pumps, sensors and other ancillary devices directly from the CelliGen 310 bioreactor. Some bioreactor´s characteristics are:

  • Available 5.0 L culture,
  • Batch, fed-batch & continuous modes for growing high-density cultures of mammalian, insect & plant cells
  • For secreted products, a patented packed-bed basket option is available to maximize cell productivity regardless of cell type
  • Fully-integrated system is ready for out-of-the box startup.
  • Mass flow controller with 4-gas control. Vessel, pH/DO/and level/foam probes, hoses are present.

BelloCell 3000 Bioreactor  (Eukaryotic culture)
The eucaryote cell culture system BelloCell 3000 provides a protected, controlled, and contained environment for the growth of cell cultures. Maximal capacity is equivalent to extremely high yields — averaging 2.4 x 1010cells for a system with four bottles. The BelloCell 3000 consists of three major components; a control box, the BelloStage unit, and ready-to-use disposable 500 ml bottles. The BelloStage unit, which holds up to four disposable cell culture bottles, moves the bottles’ contents up and down according to your program, using a platform to compress and expand the bellows built into each bottle, to optimize oxygenation. As the platform lifts, it compresses the bellows, sending the media into the chamber that contains the BioNOC II® disks; as the platform descends, the media returns to the expanding bellows, exposing the carrier disks to the atmospheric environment. The growth cells in these carriers grow along the fibers, and then pile up to fill the space in the net.

EmulsiFlex-C5 Homogenizer

The EmulsiFlex-C5 has an air/gas driven, single-acting, high-pressure pump. Quiet operation is due to a specially designed pump motor pilot valve.
The EmulsiFlex-C5 has a capacity of 1-5L/hr. The flow rate depends upon the selected homogenizing pressure. Samples, as small as 7mL, can be processed with a hold back volume of less than 1mL. AVESTIN provides stainless steel heat exchangers to control inlet and outlet temperatures. The entire EmulsiFlex-C5 can be immersed in a water bath for temperature control. The pressure is adjustable between 500-30,000psi (30-2,000bar), which is high enough for virtually every homogenization application.
The equipment is Steam-In-Place (SIP) sterilizable. It is suitable for clean room and GMP manufacturing. All wetted parts are autoclavable. For inspection, all wetted parts can be disassembled and reassembled in a short time.

Multitron 2 Incubated Shaker

The Multitron II is a large capacity shaking incubator which combines flexibility and operational safety with the optimum utilization of space by its modular construction. UPR laboratory have three units with culture capacity of 5 liters each one for differents procariotyc cultures. In addition integral microprocessor offers a wide variety of control possibilities, including high temperature range (Cº 12 to Cº 65), oxygenation control and light intensity.

services

SERVICES

Services that are currently being provided
1.    Protein expression in prokaryotic and eukaryotic systems:

  • E. coli expression
  • Baculovirus system
  • Mammalian cells expression
  • Drosophila expression system

2.     Optimizing conditions for the expression of Recombinant Proteins
3.     Refolding and soluble production of Recombinant Proteins (RP)
4.     Maintaining collections of expression vectors and bacterial strains
5.     Developing and testing new vectors and protocols

RESEARCH

Recombinant proteins, have demonstrated a high impact in basic research as well as in the biomedical field. However, in many cases obtaining a soluble and homogenous product is not possible, limiting their applications. Several strategies were developed over the last decades to overpass these limitations. In this regard, our group had generated a vector suite that facilitates the cloning steps and allows the evaluation of several parameters that can improve the soluble expression of a target protein. At the moment the vector suite is used not only in our group, but by several groups from the IPMONT and from laboratories from Argentina, France, USA, Sweden and India among others (Correa et al., Front Microbiol., 2014; Correa et al., Biotechnol J 2011; Correa et al., Methods Mol Biol 2015).

In the context of therapeutics tools related with cancer, our group is recently focused on the generation artificial binding proteins know as Affitins. This class of  proteins, present a broad range of advantages when compared with classical therapeutics antibodies, that could be taken into account in the development of therapeutic approaches. Compared with classical therapeutics antibodies Affitins are able to maintain high affinity constants even when their molecular weight remains small. This could be very useful in lymphoid neoplasms, in order to gain access into solid tissues as secondary lymphoid organs, where leukemic cells receive pro-survival signals acquiring favorable proliferative conditions. In this line, a new generation of combinatorial protein engineering technologies has been recently set up in our laboratory. The results in this line has been allowed to propose the use of these artificial binding proteins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general, that could be envisaged for futures tumor therapy strategies (Correa et al., Plos One, 2014).

 

EDUCATION-COURSES

  1. “Introduction to structural and functional analysis of proteins,” Coordinators: Agustin Correa,Horacio Botti,Matias Machado,Felipe Trajtenberg,Lucia Turell, Bruno Manta . Institut Pasteur de Montevideo. Setiembre-Noviembre 2014.
  2. Curso de postgrado: “Expression, Purification and Crystallization of Recombinant Proteins by High-throughput Methodologies”. Coordinadores: Pablo Oppezzo y Renaud Vincentelli. Febrero 2013.
  3. Curso de postgrado: “Expresión de Proteínas Recombinantes”. PEDECIBA- Biología- Maestría en Biotecnología, Facultad de Ciencias – Institut Pasteur de Montevideo.  Coordinadores: Oppezzo P. (IP Montevideo) y Marín M. (FC, UdelaR) – 5 de noviembre – 10 de diciembre de 2008.

 

GRANTS

  •  Fondo María Viña – Dr. Pablo Oppezzo – “Development of Artificial Binding Proteins (Affitins) to evaluate new prognosis and treatment strategies in Chronic Lymphocytic Leukemia”– 2015-2017 – ANII, Uruguay
  • CSIC, I+D2014 – Dr. Pablo Oppezzo – “Implicancias de la expresión anómala de la enzima mutagénica  AID en la progresión de la Leucemia Linfoide Crónica” – 2014-2017 –Comisión sectorial de investigación científica de la Universidad de la República, Uruguay.
  • Fondo Clemente Estable – Dra. Cecilia Abreu – “Estudios genómicos del perfil de metilación del ADN en una población tumoral leucémica sobre-expresando la enzima AID” – 2013-2014 – ANII, Uruguay.
  • Fondo Clemente Estable – Dr. Pablo Oppezzo – “Implicancias de la expresión anómala de la enzima mutagénica AID en los procesos leucémicos: Desarrollo de un modelo tumoral” – 2013-2015 – ANII, Uruguay
  • Fondo María Viñas – Dr. Pablo Oppezzo – “Expresión de la Lipoproteína Lipasa en las células B de la Leucemia Linfoide Crónica (LLC): Hacia el desarrollo de un nuevo marcador pronóstico” – 2013-2015 – ANII, Uruguay
  • Fondo CYTED – Dr. Pablo Oppezzo – “Red-iberoamericana de Leucemia Linfoide Crónica: hacia el desarrollo de nuevos marcadores pronósticos” – 2011-2014 – CYTED.
  • Proyectos Transversales IPMont – Dr. Pablo Oppezzo –. “Genomic landscape of the methylation pattern and the microRNAs/mRNAs expression in progressive patients with Chronic Lymphocytic Leukemia” – 2013-2014 – Institut Pasteur de Montevideo, Uruguay.
  • Fondo Lady Tata – Dr. Pablo Oppezzo – “Characterisation of the proliferating pool in CLL.  Is AID expression a marker of this subpopulation?” – 2008-2011 – Lady Tata Foundation, United Kingdom.

 

PUBLICATIONS

  1. Correa A, Oppezzo P. Overcoming the solubility problem in E. coli: available approaches for recombinant protein production. Methods Mol Biol. 2015;1258:27-44. doi: 10.1007/978-1-4939-2205-5_2.
  2. Correa A, Ortega C, Obal G, Alzari P, Vincentelli R, Oppezzo P. Generation of a vector suite for protein solubility screening. Front Microbiol. 2014 Feb 25. eCollection 2014.
  3. Alem, D.; Diaz, P.; Leoni, C.; De Simone, S.G.; Correa, A.; Oppezzo, P.; Dalla Rizza, M. In Search of Topical Agricultural Biofungicides: Properties of the Recombinant Antimicrobial Peptide TrxAq-AMP obtained from Amaranthus quitensis. Journal of Microbial & Biochemical Technology, 2014, v. 6, no.5, p. 268-273.
  4. Correa A, Pacheco S, Mechaly AE, Obal G, Béhar G, Mouratou B, Oppezzo P, Alzari PM, Pecorari F. Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins). PLoS One. 2014 May 13;9(5) eCollection 2014.
  5. Correa A, Trajtenberg F, Obal G, Pritsch O, Dighiero G, Oppezzo P, Buschiazzo A. Structure of a human IgA1 Fab fragment at 1.55 A resolution: potential effect of the constant domains on antigen-affinity modulation. Acta Crystallogr D Biol Crystallogr 2013 Mar;69(Pt 3):388-97. doi: 10.1107/S0907444912048664.
  6. Oppezzo P, Obal G, Baraibar MA, Pritsch O, Alzari PM, Buschiazzo A. Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: the carbohydrate binding mechanism involves residual sialidase. Biochim Biophys Acta. 2011 Sep; 1814(9):1154-61. doi:10.1016/j.bbapap.2011.04.012.
  7. Correa A, Oppezzo P. Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J. 2011 Jun;6(6):715-30. doi: 10.1002/biot.201100025. Review.
  8. Moratorio G, Obal G, Dubra A, Correa A, Bianchi S, Buschiazzo A, Cristina J, Pritsch O. Phylogenetic analysis of bovine leukemia viruses isolated in South America reveals diversification in seven distinct genotypes. Arch Virol. 2010 Apr;155(4):481-9. doi: 10.1007/s00705-010-0606-3.

CONTACT

Dr Pablo Oppezzo

e-mail: poppezzo@pasteur.edu.uy