Protein Purification: Isolating Fluorescent Protein from Bacteria

Protein Purification Lab Introduction
This lab follows either “Bacterial Transformation” or “Bacterial Transformation with Gene Regulation.”

Obtaining purified protein from a genetically modified organism (GMO) is an important step for many pharmaceutical companies and research labs. Whether manufacturing proteins for clinical use, such as insulin, or studying the function of proteins in biochemical assays, protein purification is an essential process.
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    In this lab, students will use nickel affinity chromatography to isolate fluorescent proteins from E. coli bacteria that they have previously transformed with the pKiwi plasmid (or another BABEC plasmid).

    Nickel affinity chromatography utilizes six histidine (his) amino acids that have been added to the recombinant green fluorescent protein as an artificial “tag.” This 6XHis tag has a high affinity for nickel resin. Since only the recombinant protein will possess this unique tag, the purification process can specifically separate FPs from the other proteins in the cell lysate.

    This lab provides an opportunity for hands on exploration of the applications of genetic engineering. It can introduce or review topics ranging from recombinant DNA, transcription, translation, protein expression, cell lysis and clearly demonstrates how proteins can be isolated for use in research or medicine.

Lesson Resources
Student Guide – Download and edit student worksheets to be completed during the Protein Purification Lab.
Lesson Slides – Articulates with the student guide above and accompanying lesson plans below.
Lesson Plan – Suggested times for pacing, sequence and activities for the lab. Includes vocabulary tool and other lesson resources.
Supplemental Resources – Includes background information, outside articles, videos, and more related to the lab

Lab Resources
Student Protocol – Students follow these instructions when doing the Protein Purification Lab.
Preparing the Classroom for the Lab – How to set up group stations for this lab experience.
Inventory Guide – Reagents and materials checklist for one classroom kit. Includes equipment and classroom needs for the lab.

  • Isolate purified Green Fluorescent Protein from successfully transformed E. coli bacteria
  • Describe the process of protein purification using nickel-affinity chromatography
  • Create a scientific model that explains how to isolate and utilize a protein product from a genetically modified organism

Disciplinary Core Ideas:
LS1-A Structure and Function

  • “…genes are regions in the DNA that contain the instructions that code for the formation of proteins, which carry out most of the work of cells.”

LS3-A Inheritance of Traits

  • “…genes (expressed) by the cell may be regulated in different ways…”

Science and Engineering Practices:
Developing and Using Models
Constructing Explanations

  • Students explain how to isolate and utilize a protein product from a genetically modified organism by creating a graphic model.

Crosscutting Concepts:
Systems and System Models

  • Isolating fluorescent protein from transformed bacteria is a model for protein purification in other applications such as medicine and food production.

Familiarity with the following biology concepts may be helpful for students prior to using the lesson resources

  • Structure and function of DNA and proteins
  • Relationship between genes and the proteins they code for (DNA → mRNA → protein)
  • Process of bacterial transformation with pKiwi or another plasmid containing a fluorescent protein-coding gene

Kitty MeiProtein Purification: Isolating Fluorescent Protein from Bacteria