Question

Calculations for the Purification of a Recombinant Protein The purification of a recombinant protein is carried out starting with 100 liters of a clarified cell lysate (i.e., the cells have been lysed, and the cell debris has been removed to give a clarified solution), which has a total protein concentration of \(0.36 \mathrm{mg} / \mathrm{ml}\) and a recombinant protein concentration of \(2.2 \mathrm{U} / \mathrm{ml}\), where U denotes units of biological activity of the recombinant protein. It is known that the completely pure recombinant protein has a specific activity of \(40.0 \mathrm{U} / \mathrm{mg}\). Purification is continued until a chromatography step that yields \(2.0\) liters of a fraction containing the protein, with a total protein concentration of \(1.11 \mathrm{mg} / \mathrm{ml}\) and a recombinant protein concentration of \(43.2 \mathrm{U} / \mathrm{ml}\).For the recombinant protein, calculate the starting and ending purity, the starting and ending specific activity, and the percentage yield and fold purification through the chromatography step.

Short answer

The initial purity is 6.11, and the final purity is 38.92. The initial specific activity is 2.2, and the final specific activity is 43.2. The yield is 39.27%, and the fold purification is 6.37 times.

Step-by-step solution

Calculate initial and final amounts of total and recombinant protein

First, the total initial amount of protein is calculated by multiplying the initial volume by the initial protein concentration, making sure to convert liters to milliliters. The same operation is done to find the total initial recombinant protein activity, but instead multiplied by the recombinant protein activity concentration. Repeat these calculations with the final volume and concentrations respectively. Initial total protein = \(100 \times 1000 \times 0.36 = 36000\) mg, Initial recombinant protein = \(100 \times 1000 \times 2.2 = 220000\) Units, Final total protein = \(2 \times 1000 \times 1.11 = 2220\) mg, Final recombinant protein = \(2 \times 1000 \times 43.2 = 86400\) Units.

Calculate initial and final purity and specific activity

Purity is the fraction of the total protein that is recombinant protein, and specific activity is units of biological activity per milligram of protein. Therefore, initial purity = \(220000/36000 = 6.11\) Units/mg, final purity = \(86400/2220 = 38.92\) Units/mg, initial specific activity = \(2.2\) Units/mg, and final specific activity = \(43.2\) Units/mg.

Calculate yield and fold purification

Yield is the fraction of the initial recombinant protein activity that is present after purification, and fold purification is the increase in purity after purification. Therefore, yield = \(86400/220000 \times 100 = 39.27\%\), and fold purification = \(38.92/6.11 = 6.37\) fold.

Key concepts

Recombinant Protein Purification

Recombinant protein purification is a vital process in biotechnology where specific proteins, engineered through recombinant DNA technology, are isolated from a complex mixture. The goal is to achieve a high degree of purity while retaining as much biological activity as possible.

This process often begins with lysing cells to release their contents, followed by removing cell debris to obtain a clarified solution. The protein of interest is then concentrated and separated from other proteins and impurities. The challenges in purification include:

• Maintaining the protein's stability throughout the process.
• Maximizing yield while minimizing costs.
• Ensuring the purified protein retains its biological functionality.

By computing the starting and ending purity, we understand how the concentration of the target protein changes through the process, which is crucial for evaluating the effectiveness of the purification strategy.

Chromatography in Biotechnology

Chromatography is a key method used in biotechnology for the separation and purification of biomolecules such as proteins. It involves passing a mixture dissolved in a "mobile phase" through a "stationary phase," which separates the mixture into its components.

There are various types of chromatography, each suited for different purposes:

• Size-Exclusion Chromatography: Separates molecules based on size, with larger molecules eluting faster than smaller ones.
• Ion-Exchange Chromatography: Separates based on the charge of molecules, where ions are attracted to opposite charges on the resin.
• Affinity Chromatography: Utilizes the specific interaction between a binding pair (e.g., enzyme and substrate).

Each method has its advantages and limitations, and the choice depends on factors such as the target protein's properties and the level of purity required. Chromatography can greatly enhance purification efficiency, resulting in higher yields of the desired protein.

Specific Activity Calculations

Specific activity is a crucial metric in bioprocess engineering, especially when purifying proteins. It refers to the activity of an enzyme per milligram of total protein, serving as an indicator of enzyme purity and activity. The specific activity calculation helps assess how effectively a protein maintains function after purification.

Here's how specific activity is calculated:

• Calculate the total protein concentrations and the total activity (in units) before and after the purification step.
• Divide the total activity by the total protein concentration for both initial and final samples.

Initially, the recombinant protein has a specific activity of a certain value, showing how active it is compared to other proteins in the mixture. After purification, you calculate the new specific activity to gauge if the desired protein's purity and functionality have improved. High specific activity in the final product reflects successful purification and a high proportion of the target protein compared to contaminants.