Proteomics of Lactococcus lactis: An Overview

Mark Wilkins and colleagues coined the terms proteome and proteomics in the early 1990s.

The proteome is the complement protein or complete set of proteins expressed by the genome of an organism and is present in a single cell in a particular environment. It differs from cell to cell and varies through time.

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What is Proteomics?

  • Proteomics, a new type of omics, is the study of the proteome. 
  • The study of the protein composition, structure, function, interaction, and cellular activities is known as proteomics.
  • It mainly focuses on the organism’s structure and function than the genomics. 
  • The protein expression varies with time and environmental conditions, which makes it more complicated than genomics. 
  • Moreover, it is a multi-step technique, so it controls every step in the process to avoid non-biological factors interfering with protein expression and interaction.
  • The two classifications of proteomics are protein expression mapping and protein interaction mapping.

Techniques of Proteomics

There is a wide range of proteomics techniques. They are:

  1. One-dimensional (1D) and Two-dimensional (2D) gel electrophoresis (2-DE)) for protein separation.
  2. Gel-free high-throughput screening techniques include multidimensional protein identification technology, isobaric tagging for relative and absolute quantitation (iTRAQ), stable isotope labeling with amino acids in cell culture (SILAC), and an isotope-coded affinity tag(ICAT).
  3. Antibodies-based methods, such as ELISA (Enzyme-linked immunosorbent assay), immunoprecipitation, immune-electrophoresis, and Western blot.
  4. Chromatographic methods include ion exchange, size exclusion, and affinity chromatography. HPLC (High-Performance liquid chromatography) is a technique for analytical proteomics.
  5. PAGE, Mass spectrometry methods
  6. X-ray crystallography and nuclear magnetic resonance spectroscopy 
  7. Analytical, functional, and reverse phase microarrays.
Proteomics Workflow and Techniques
Proteomics Workflow and Techniques.

Objectives of Proteomics

a. It helps in the separation of protein/ peptide.

b. It helps to identify and characterize the resolved protein by mass spectrometry (MS).

c. Proteomics helps to analyze protein expression at different levels, such as post-transcriptional, transcriptomic, and genomic levels, both qualitatively and quantitatively. 

d. It helps in the analysis and application purposes.

e. It helps in the movement of protein between subcellular compartments.

Types of Proteomics

  1. Structural Proteomics:
  • It involves building a body of structural information that predicts probable structure and potential function. 
  • The techniques used in structural proteomics are Nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the three-dimensional structure and structural complexities of functional proteins. 
  • The nuclear pore complex study is one of the examples of structural proteomics.
  1. Functional proteomics:
  • It involves studying the protein functions and analyzing the characteristics of molecular protein networks (protein partner’s interactions) present in a living cell through proteomics techniques.
  1. Expression proteomics:
  • Expression proteomics involves the study of quantitative and qualitative expression of proteins between samples. 
  • It specifies protein expression that differs between two conditions, such as patients and controls. 
  • It helps to find specific proteins and new proteins of disease in signal transduction. It identifies the patterns of protein expression in different cells. 
  • An example is comparing tumor tissue samples to normal tissue to identify protein level differences. The detection technique is 2-DE and mass spectrometry (MS).

Introduction of Lactococcus lactis

  • Lactococcus lactis, a member of the mesophilic group, is a gram-positive, catalase-negative, facultatively anaerobic, nonmotile, and non-spore-forming bacterium with a G+C content of ∼35 mol%, which can grow at 10°C but not at 45°C. Hexose diphosphate pathway producing l (+)-lactic acid ferment glucose.
  • Lactococcus lactis, the primary organism for lactic acid bacteria (LAB), encounters three environmental stimuli such as H+, lactate, and undissociated lactic acid during fermentation. It is due to the accumulation of lactic acid (the predominant fermentation product). Lactic acid inhibits the growth of cells and accumulates essential products, such as nisin.
  • Lactic acid bacteria, non-pathogenic, are widely used in fermented food and pharmaceutical production. It is potential for new biomedical applications such as vaccine delivery, gene delivery, heterologous protein expression, and therapeutic drug delivery. Lactic acid bacteria are generally recognized as safe (GRAS).
  • It has the property of a simple mechanism, rapid growth, and lacks toxic substances.
Proteomics of Lactococcus lactis
Proteomics of Lactococcus lactis

Proteomics of Lactococcus lactis

  • Lactococcus lactis produces proteins, plasmids, and metabolites that are used in the food industry, pharmaceutical, and cosmetic areas. A KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis helps to identify the most represented metabolic pathways in wild-type L. lactis.
  • The core proteome consists of the expression of membrane proteins and enzymes, which are responsible for the cell wall synthesis (e.g., basic membrane protein A, chitinase, exodeoxyribonuclease, pyruvate carboxylase fibronectin-binding protein)- the adhesive properties of Lactococcus lactis required for mucosal vaccination mechanisms and other immunomodulation processes.
  • Lactococcus lactis is a gene-expression host for eukaryotic MPs because it has moderate proteolytic activity, lacks the formation of an inclusion body, lacks the production of endotoxin (allows bacteria and protein use in biotechnological and therapeutic applications), and its ability to efficiently target the MPs into a single plasma membrane.
  • Instead of inclusion bodies formation, Other Lactococcus lactis methods include the formation of polar clusters by the mRNAs of recalcitrant MPs, which stops cell division and leads to degradation rather than aggregation.
  • Lactococcus lactis permits the direct execution of functional studies using membrane vesicles and whole cells. Its membrane contains different lipids and is especially rich in glycolipids and cardiolipin. These lipids are absent in E. coli membranes. 
  • Lipids are crucial for the stability, conformations, and functionality of MPs. It depends on the nature and functions of the MP generated. The composition of lipids shows a positive impact on the expression and the functionality of the MP.

The Lactococcus lactis expression system produces the malaria antigens because of its properties such as:

  1. It accommodates cysteine-rich proteins.
  2. It offers a scalable fermentation process.
  3. Lactococcus lactis clone allows the recombinant protein, consist disulfide bonds, to secrete into the culture medium, thereby simplifying the purification process.
  4. It exhibits a similar codon bias as P. falciparum and, therefore, does not require codon optimization before protein expression.

L. lactis strains (L. lactis subsp. lactis NCDO2118, L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. cremoris MG1363) are applied for biotechnology applications, such as pharmaceutical-grade plasmid DNA (pDNA) production for DNA vaccination, and recombinant protein expression or metabolites for mucosal vaccination.

Detection

  • Lactococcus lactis proteome can be characterized by genomic dataset, high‐throughput proteomic, and PPI network analysis.
  • SDS-PAGE and Western Blot analysis can detect recombinant protein.

Metabolically Engineered Strategies

  1. It helps to increase the production yield of industrially essential proteins or metabolites. Gene knockouts or the heterologous enzyme expression strategy helps to increase the productivity of L. lactis for compounds such as alanine (used as a food sweetener and for pharmaceutical applications) or diacetyl (used in many dairy products as well as in the wine industry).
  2. It also increases the production of pharmaceutically valuable compounds, such as folate (vitamin B11), riboflavin (vitamin B2), and hyaluronic acid (polysaccharide with medical applications).
  3. It also helps to produce recombinant protein by the changes feature, such as the deletion of nonessential DNA regions by using the Cre-loxP deletion system, turning it into a faster-growing strain with a higher biomass yield, a higher ATP content, and less maintenance demands.

References

  1. Al-Amrani, S., Al-Jabri, Z., Al-Zaabi, A., Alshekaili, J., & Al-Khabori, M. (2021). Proteomics: Concepts and applications in human medicine. World Journal of Biological Chemistry, 12(5), 57–69. https://doi.org/10.4331/wjbc.v12.i5.57
  2. Chinnaswamy, S. (2015, January 13). Proteomics ppt [Slide show]. SlideShare. https://www.slideshare.net/ShashikalaChinnaswam/proteomics-ppt
  3. Embl-Ebi. (n.d.). What is proteomics? | Proteomics. https://www.ebi.ac.uk/training/online/courses/proteomics-an-introduction/what-is-proteomics/
  4. proteomics. (2019, February 20). [Slide show]. SlideShare. https://www.slideshare.net/vruddhidesai/proteomics-132537596
  5. Mills, S., Ross, R., & Coffey, A. (2011). Lactic Acid Bacteria | Lactococcus lactis. In Elsevier eBooks (pp. 132–137). https://doi.org/10.1016/b978-0-12-374407-4.00266-1
  6. Wu, H., Zhao, Y., Du, Y., Miao, S., Liu, J., Li, Y., Caiyin, Q., & Qiao, J. (2018). Quantitative proteomics of Lactococcus lactis F44 under cross-stress of low pH and lactate. Journal of Dairy Science, 101(8), 6872–6884. https://doi.org/10.3168/jds.2018-14594
  7. Frelet-Barrand, A. (2022). Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins. Biomolecules, 12(2), 180. https://doi.org/10.3390/biom12020180
  8. Wu, F., Xie, X., Du, T. et al. Lactococcus lactis, a bacterium with probiotic functions and pathogenicity. World J Microbiol Biotechnol 39, 325 (2023). https://doi.org/10.1007/s11274-023-03771-5
  9. Singh, S. K., Tiendrebeogo, R. W., Chourasia, B. K., Kana, I. H., Singh, S., & Theisen, M. (2018). Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum. Microbial Cell Factories, 17(1). https://doi.org/10.1186/s12934-018-0902-2
  10. Singh, S. K., Tiendrebeogo, R. W., Chourasia, B. K., Kana, I. H., Singh, S., & Theisen, M. (2018). Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum. Microbial Cell Factories, 17(1). https://doi.org/10.1186/s12934-018-0902-2

About Author

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Prativa Shrestha

Prativa Shrestha is an enthusiastic person pursuing a master's degree in Food Microbiology from St. Xavier's College., Kathmandu. Currently, She is doing thesis work at Nepal Academy of Science and Technology (NAST). She loves to explore new ideas and showcase her creativity. She has also published two research articles. Moreover, She is interested in research fields like Food microbiology Biotechnology, and enzyme production.

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