Proteomics: recombinant protein production, purification and characterization

This brand new Cutting Edge Biomedical Solution allows you to obtain customized recombinant proteins and a wide proteomic characterization.

Scientific Leader / leaders of the project: Neus Ferrer Miralles / Cristina Ruiz Romero
Coordinator of the project: Merce Márquez Martínez (U1)

Industrial gap/problem covered

The usability of recombinant proteins across diverse applications largely depends on their similarity to physiological counterparts. To maximize the probability of achieving a successful protein it is necessary to conduct proteomic studies for its deep characterization. These studies, among other objectives, aim to identify the purified protein, ascertain its molecular weight, and analyze post-translational modifications or putative molecular interactions.

Description

Proteomic studies using mass spectrometry play a crucial role in establishing production and purification procedures for recombinant proteins on a case-by-case basis and ensuring their quality for various applications. They provide valuable information on the identity, purity, structural integrity, consistency, reproducibility, and functional aspects of these proteins, ultimately contributing to the success of various biotechnological and biomedical applications.

Examples of CIBER BBN collaborative intramural  projects implementing this Cutting-Edge Biomedical Solution:

Recently, two collaborations have been conducted, enabling the supply of properly identified active recombinant proteins to the client.

• In one of the projects, successful purification of an active recombinant protein from Stenotrophomonas maltophilia was achieved using the E. coli expression system. Purification of this protein had not been accomplished until now. These results are pending publication. 
• In the second project, a human enzyme has been purified using the E. coli expression system for industrial purposes. It is pending of commercialization.

Both proteins were designed, produced and purified by U1, and finally characterized by U30. To preserve the confidential rights of our users, we cannot provide more details on the type of protein nor the name of the companies.

Services involved:

The U1 Protein Production Platform is responsible for designing, producing, and purifying recombinant proteins tailored to the requirements of clients. The U30 Proteomics unit performs characterizations on these recombinant proteins by mass spectrometry, typically involving their identification and, as necessary, carrying out additional analyses based on specific needs (i.e., analysis of post-translational modifications, quantitative studies, and others).

• Molecular cloning:  Cloning of the gene that codifies for the protein of interest in the chosen expression vector. U1-S01.
• Bioproduction of proteins: Selection of the optimal protein production conditions using small-scale expression and later produce the protein on a medium-scale. U1-S02.
• Proteins purification: Identification of the most appropriate strategy for purification, given the physical and chemical properties, opting either for purification modules or, in the case of fusion proteins, elements which enable purification by affinity. U1-S03.
• Sample preparation for proteomic analysis: This service includes all necessary steps to prepare samples for different proteomic analyses. In this case protein digestion (in gel or in solution) with proteolytic enzymes (trypsin, chymotrypsin, lys-C…), to obtain the correspondent peptides. U30-S01.
• Protein identification by mass spectrometry (MS) or MS/MS: Identification of proteins in biological samples, either by intact protein analysis (MALDI-TOF) or protein digestion followed by peptide mass fingerprinting (PMF, MS) or peptide fragmentation (MS/MS). U30-S03.
• Data analysis of proteomics experiments: Use of dedicated software for the analysis and interpretation of the results obtained from the proteomic analyses. It includes search engines (MaxQuant, MSFragger) for protein identification and quantification, software for statistical analysis (Perseus) and programs for integrated solutions (Peaks) that also enable the analysis of post-translational modifications (PTMs), sequence variants and mutations. U30-S09.

• U1
• U30

The PPP Unit is at the Institute of Biotechnology and Biomedicine of the Autonomous University of Barcelona (IBB-UAB). It counts with the necessary facilities for the design, production, and purification of recombinant proteins. This facility is coordinated by the Nanobiotechnology Group, led by Prof. Antonio Villaverde. It has both highly specialized personnel and the necessary equipment to offer a “tailored” service for the design, production, and purification of recombinant proteins using both prokaryotic and eukaryotic expression systems. Its location allows this service to be linked with other complementary services available within the university (Cell Culture, Cytometry, Production of Antibodies, Microscopy, Proteomic, and Bioinformatic, X-ray crystallography, and Microarray and Sequencing Services), as well as facilitating the management of subsequent uses of the produced protein.

The Proteomics Unit is at the Biomedical Research Institute of A Coruña (INIBIC-CHUAC-UDC). It counts with all necessary facilities for the analysis of proteins at a small- and large-scale, including three mass spectrometers, liquid chromatographs, electrophoresis systems and instruments for immunoassay development and readout. The Platform is fully equipped to perform high resolution shotgun proteomics experiments, using isotopic labeling strategies or label free quantification (LFQ) strategies and employing either data-dependent (DDA) or data-independent (DIA) acquisition methods. The services of the Unit involve all steps of this analysis, from sample preparation to data analysis, reporting and submission of results. The Unit personnel are highly specialized and have a vast experience offering tailored services to aid users from the experimental design step. The Unit has also infrastructure for targeted proteomics experiments for the quantification of specific protein panels by the Multiple Reaction Monitoring (MRM) mass spectrometry technique. Finally, the Proteomics Unit has the equipment and expertise for the development and readout of ELISA or Luminex-based immunoassays, focusing on smaller numbers of protein targets.