Acta Scientific Microbiology (ISSN: 2581-3226)

Review Article Volume 4 Issue 11

Cloning, Expression, and Purification of Recombinant Protein Utilizing Bacterial Expression System

Ansab Akhtar*

Assistant Professor, Chandigarh College of Pharmacy, Chandigarh Group of Colleges, Landran, Mohali, Punjab, 140307, India

*Corresponding Author: Ansab Akhtar, Assistant Professor, Chandigarh College of Pharmacy, Chandigarh Group of Colleges, Landran, Mohali, Punjab, 140307, India.

Received: September 25, 2021 ; Published: October 27, 2021



Background: The primary interest for most molecular biologists has remained genes. The isolation and amplification of genes are the foremost criteria for gene-related studies.

Main Body: A simple strategy for the same is gene cloning by incorporating a gene into a vector or vehicle carrying a DNA molecule. This is further taken through the replication process with the help of living cells. A recombinant DNA molecule comes into the picture as a result of combining two DNAs possessing disparate sources. The required gene isolated from any source can be taken to the cloning process by inserting it into a suitable vector with desired properties to create recombinant DNA. Cloning involves the use of restriction enzymes termed restriction endonucleases or molecular scissors isolated from various sources. Various other enzymes like DNA ligases, used for joining the DNA fragments, DNA polymerases, used in producing multiple replicas of a source DNA are involved in cloning procedures. Furthermore, the produced recombinant DNA can be introduced into the host system in order to synthesize the product, the gene codes for. The obtained protein product is finally concentrated and purified by affinity chromatography, followed by analysis and confirmation by western blotting.

Conclusion: Cloning of genes and expressing the recombinant protein in bacterial system followed by purification by chromatography involves important tools and techniques in molecular biology.

Keywords: DNA Insertion; Bacterial Expression System; Molecular Cloning; Recombinant DNA Technology; Affinity Chromatography; Western Blotting



  1. Moriya H. “Quantitative nature of overexpression experiments”. Molecular Biology of the Cell 26 (2015): 3932-3939.
  2. Sheehan D and O’Sullivan S. “Fast protein liquid chromatography”. Protein Purification Protocols: Springer (2004): 253-258.
  3. Garibyan L and Avashia N. “Research techniques made simple: polymerase chain reaction (PCR)”. The Journal of Investigative Dermatology 133 (2013): e6.
  4. Kramer MF and Coen DM. “Enzymatic amplification of DNA by PCR: standard procedures and optimization”. Current Protocols in Molecular Biology 56 (2001): 15.1.1-.1.4.
  5. Xu W., et al. “A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis”. PloS one 7 (2012): e22900.
  6. Sanderson BA., et al. “Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis”. Analytical Biochemistry 454 (2014): 44-52.
  7. Yılmaz M., et al. “Principles of nucleic acid separation by agarose gel electrophoresis”. Gel Electrophoresis–Principles and Basics 4 (2012): 33.
  8. Kasibhatla S., et al. “Analysis of DNA fragmentation using agarose gel electrophoresis”. Cold Spring Harbor Protocols 2006 (2006): pdb. prot4429.
  9. Downey N. “Extraction of DNA from agarose gels”. E coli plasmid vectors: Springer (2003): 137-139.
  10. Loenen WA., et al. “Highlights of the DNA cutters: a short history of the restriction enzymes”. Nucleic Acids Research 42 (2014): 3-19.
  11. Wang S., et al. “A new strategy for species identification of planktonic larvae: PCR–RFLP analysis of the internal transcribed spacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC”. Journal of Plankton Research 28 (20065): 375-384.
  12. Scheich C., et al. “An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography”. BMC Biotechnology 3 (2003): 1-8.
  13. VAASUDEVAN A. “Expression and characterization of FAT1 and atrophin 1 proteins regulating planar cell polarity and MBD1 protein involved in lymphoma” (2009).
  14. Stuchbury G and Münch G. “Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA”. Cytotechnology 62 (2010): 189-194.
  15. Tomkinson AE., et al. “DNA ligases: structure, reaction mechanism, and function”. Chemical Reviews 106 (2006): 687-699.
  16. Gansauge M-T., et al. “Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase”. Nucleic Acids Research 45 (2017): e79-79e.
  17. Cranenburgh R. “An equation for calculating the volumetric ratios required in a ligation reaction”. Applied Microbiology and Biotechnology 65 (2004): 200-202.
  18. Chang AY., et al. “Preparation of calcium competent Escherichia coli and heat-shock transformation”. JEMI Methods 1 (2017): 22-25.
  19. Green R and Rogers EJ. “Transformation of chemically competent E. coli”. Methods Enzymology 529 (2013): 329-336.
  20. Asif A., et al. “Revisiting the mechanisms involved in calcium chloride induced bacterial transformation”. Frontiers in Microbiology 8 (2017): 2169.
  21. Sezonov G., et al. “Escherichia coli physiology in Luria-Bertani broth”. Journal of Bacteriology 189 (2007): 8746-8749.
  22. Slager J., et al. “Antibiotic-induced replication stress triggers bacterial competence by increasing gene dosage near the origin”. Cell 157 (2014): 395-406.
  23. Froger A and Hall JE. “Transformation of plasmid DNA into E. coli using the heat shock method”. Journal of Visualized Experiments: JoVE (2007).
  24. Chhetri G., et al. “An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli”. MethodsX 2 (2015): 385-391.
  25. Iacoviello MP., et al. “Sterile preparation of antibiotic-selective LB agar plates using a microwave oven”. BioTechniques 30 (2001): 963-965.
  26. Unger T., et al. “Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression”. Journal of Structural Biology 172 (2010): 34-44.
  27. Agrawal V and Roy N. “Contaminating insert degradation by preincubation colony PCR: A method for avoiding false positives in transformant screening”. Analytical Biochemistry 375 (2008): 159-161.
  28. Zhang S and Cahalan MD. “Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit”. JoVE (Journal of Visualized Experiments) (2007): e247.
  29. Li J-F., et al. “Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology”. Plant Methods 6 (2010): 1-8.
  30. Ehrt S and Schnappinger D. “Isolation of plasmids from E. coli by alkaline lysis”. E coli Plasmid Vectors: Springer (2003): 75-78.
  31. Li X., et al. “A continuous process to extract plasmid DNA based on alkaline lysis”. Nature Protocols 3 (2008): 176.
  32. Petryszak R., et al. “Expression Atlas update—an integrated database of gene and protein expression in humans, animals and plants”. Nucleic Acids Research 44 (2016): D746-D52.
  33. Segal E., et al. “Discovering molecular pathways from protein interaction and gene expression data”. Bioinformatics 19 (2003): i264-i72.
  34. Chen R. “Bacterial expression systems for recombinant protein production: E. coli and beyond”. Biotechnology Advances 30 (2012): 1102-1107.
  35. Kielkopf CL., et al. “Expression of cloned genes in E. coli using IPTG-inducible promoters”. Cold Spring Harbor Protocols 2021 (2021): pdb. prot102137.
  36. Hogwood CE., et al. “Host cell protein dynamics in recombinant CHO cells: impacts from harvest to purification and beyond”. Bioengineered 4 (2013): 288-291.
  37. Janson J-C. “Protein purification: principles, high resolution methods, and applications”. John Wiley and Sons (2012).
  38. Peach M., et al. “Solubilization of proteins: the importance of lysis buffer choice”. Western Blotting: Springer (2015): 49-60.
  39. Shrestha P., et al. “Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing”. Biotechniques 53 (2012): 163-174.
  40. Dennison C. “A guide to protein isolation”. Springer Science and Business Media (2013).
  41. Coskun O. “Separation techniques: chromatography”. Northern clinics of Istanbul 3 (2016): 156.
  42. Kielkopf CL., et al. “Purification of fusion proteins by affinity chromatography on glutathione resin”. Cold Spring Harbor Protocols 2020 (2020): pdb. prot102202.
  43. Schäfer F., et al. “Purification of GST-tagged proteins”. Methods in Enzymology 559 (2015): 127-139.
  44. Brunelle JL and Green R. “One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE)”. Methods in Enzymology 541 (2014): 151-159.
  45. Magdeldin S., et al. “Basics and recent advances of two dimensional-polyacrylamide gel electrophoresis”. Clinical Proteomics 11 (2014): 16.
  46. Sharma N., et al. “Distinction between glycomacropeptide and β-lactoglobulin with ‘stains all’dye on tricine SDS-PAGE gels”. Food Chemistry 340 (2021): 127923.
  47. Bass JJ., et al. “An overview of technical considerations for Western blotting applications to physiological research”. Scandinavian Journal of Medicine and Science in Sports 27 (2017): 4-25.
  48. Mahmood T and Yang P-C. “Western blot: technique, theory, and trouble shooting”. North American Journal of Medical Sciences 4 (2012): 429.
  49. Mruk DD and Cheng CY. “Enhanced chemiluminescence (ECL) for routine immunoblotting: an inexpensive alternative to commercially available kits”. Spermatogenesis 1 (2011): 121-122.
  50. Kendrick N., et al. “Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence”. PloS One 15 (2020): e0234645.


Citation: Ansab Akhtar. “Cloning, Expression, and Purification of Recombinant Protein Utilizing Bacterial Expression System”. Acta Scientific Microbiology 4.11 (2021): 82-94.


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