Protein pipeline

Protein pipeline

Proteins, the building blocks of life

Marine biodiversity provides access to a wealth of new proteins adapted to the unique environmental conditions marine organisms tolerate. Although many microorganisms are unsuitable for culture bioinformatics approaches can be used to decipher the unique proteins encoded in the genome.  To fully exploit the potential and value of marine proteins the development of prediction tools and tools for production of recombinant proteins, their purification, structural characterization and bioactivity are essential. The EMBRIC protein platforms bring together 3 accessible platforms with expertise in this area.

Access Providers provide expertise in bioinformatics to deduce proteins, their structural characteristics and to model protein structure. Hands on cloning into a range of vector systems and hosts is offered (prokaryote and eukaryote) and coupled to protein production and purification platforms. USTAN and UPMC-SBR offer specific expertise and facilities for protein crystallography and related analysis. UPMC-SBR have high expertise in expressing glycoproteins and fucoids. USTAN has specific expertise in pathway reconstitution and membrane proteins. CCMAR brings specific expertise in bioassays and biosensors for screening protein function.

 

 

Bioinformatics

Bioinformatics is used by access providers to isolate gene sequences from datasets and predict the amino acid sequence of proteins. The main domains in proteins can be deduced as well as the biochemical characteristics of proteins. Capacities in protein modeling also exist (I-TASSER). Additional bioinformatics applications are used to design and optimize gene constructs for protein expression in bacteria and yeast expression systems.  Open source bioinformatics programs are used such as NCBI. Access can be direct on site or by consultation with expert platforms. Depending on task complexity this can take from a day to a week to perform.

Access Providers available for this pipeline element:

 

Cloning

Gene cloning capacities exist in all the platform. Custom designed constructs can be established to optimize protein expression and subsequent purification. A range of vector systems are available and are provided on request from each service provider or by consulting their respective websites. Cloning vectors are available for different expression hosts (prokaryotes and eukaryotes) and can include a tag sequence (eg. His-, GST etc) for affinity purification. Cloning vectors containing larger tags for production of fusion proteins to increase protein size and improve protein expression and solubility are also available. Standard and custom designed solutions for expression cloning are available on consultation of access providers. The minimum time for this task with construct quality control takes 5 days.

Access Providers available for this pipeline element:

 

Expression and Purification

Successful vector constructs are expressed in prokaryote (all access providers) or eukaryote (USTAN) host systems and a range of expression hosts are held by access providers and can be selected based on need and in consultation with the access providers. Cultures for protein expression can be at various dimensions from small scale (ml) to large scale (Liters) depending on need, but will always start with small scale optimization studies to identify the most appropriate conditions. Protein purification can be small scale, larger scale and with more or less automization (all access providers have semi-automated, medium throughout purification systems) and will always start with small scale optimization studies. Production scale and degree of protein purity and therefore the approach will be determined on a project-by-project basis and this will depend on the subsequent applications of the purified protein. The minimum time for this process is two weeks but it can take months depending on the protein target.

Access Providers available for this pipeline element:

 

Functional Analysis

A range of in vitro acellular functional assays (enzyme activity, protein:protein interactions) are available for protein characterization using various instrumentation including Isothermal titration calorimetry (ITC), Biosensors such as Quartz Crystal Microbalance (QCM); Immunoassays (Western-blot, ELISA, radioimmunoassay). In vitro cell-based functional assays are also available to assess basic functions such as proliferation, apoptosis, wound healing. Whole organism screening with invertebrates. Functional screening projects require prior consultation with access providers. The minimum time for this process is two weeks but it can take months depending on the protein target and the breadth of the functional screening.

Access Providers available for this pipeline element:

 

Structural Analysis

A range of approaches are available for structural determination. That include approaches for secondary structure, interactions with other molecules and crystallography. Available equipment for structural elucidation includes CD spectroscopy, Thermofluor screens, Fluorescence emission spectroscopy and crystallography (USTAN and UPMC-SBR). The time for these projects can take from weeks to months depending on the protein target and so structural projects are more suitable as a service or as a collaborative project.

Access Providers available for this pipeline element:


Publications

L. Anjos, I. Morgado, M. Guerreiro, J.C.R. Cardoso, E. P. Melo, & D. M. Power, “Cartilage acidic protein 1, a new member of the betapropeller protein family with amyloid propensity”, PROTEINS: Structure Function and Bioinformatics, Wiley, November 2016. URL:
http://dx.doi.org/10.1002/prot.25210

Juárez, JF, Liu, H, Zamarro, MT, McMahon, S, Liu, H, Naismith, J, Eberlein, C, Boll, M, Carmona, M & Diaz, E 2015, 'Unraveling the specific regulation of the mdb central pathway for the anaerobic degradation of 3-methylbenzoate' Journal of Biological Chemistry, vol 290, pp. 12165-12183. DOI: 10.1074/jbc.M115.637074

Groisillier, A., Hervé, C., Jeudy, A., Rebuffet, E., Pluchon, P. F., Chevelot, Y., Flament, D., Geslin, C., Morgado, I. Power, D. M., Branno, M.,Moreau, H., Michel, G., Boyen, C. and Czjzek, M. 2010. Marine-Express: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms. Microbial Cell Factories. 9, 45.

 

Contact person for questions and inquiries

Deborah Power
Centro de Ciências do Mar (CCMAR)
Universidade do Algarve
tel: +351 (0)289 800 051
email: dpower@ualg.pt
www.ccmar.ualg.pt

 


Back to Research services and Discovery pipelines