17
UNIT 2
EXTRACTION OF PROTEIN FROM CELLS
Introduction:
Protein Production: An Industry Overview
Protein biochemists in the biotechnology industry like to point out that while the molecular biologists can be credited with keeping new product lines in the pipelines with their gene discovery and manipulations, it’s the protein biochemists who are paying the bills. What are they referring to? Most production facilities in the biotechnology industry are producing some sort of protein product. So, the teams of research and development scientists and engineers working for years to develop a product are usually working towards a protein production process. It is estimated that the total worldwide sales of protein products exceeds $60 billion in sales in an industry that continues to expand every year. What are these protein products? A wide variety of proteins find industrial application. These include enzymes, antibodies, hormones, blood factors, growth factors and diagnostics. The protein products used for medical diagnosis or therapies are the high dollar products. Some examples are listed below.
Table 2.1 Biopharmaceutical protein products approved for general medical use in the EU and/or USA by 2002
Product type / Examples / Number of approved productsBlood factors / Factors VIII and IX (for treating hemophilia) / 7
Thrombolytic agents / Tissue plasminogen activator or tPA (for treating heart attacks and strokes) / 6
Hormones / Insulin (for treating diabetes mellitis), growth hormones (for treating cancer and AIDS) / 28
Hemapoietic growth factors / Erythropoietin (for treating anemias), colony stimulating factors (for treating immunosuppression) / 7
Interferons / Interferons-a,-b,-g (for treating cancer, AIDS, allergies, asthma, arthritis and infectious diseases / 15
Interleukin-based products / Interleukin-2 (for treatment of cancer, AIDS, and bone marrow suppression) / 3
Vaccines / Hepatitis B surface antigen, herpes surface antigen / 20
Monoclonal antibodies / Various uses. Treatment of cancer and rheumatoid arthritis. Used for diagnostic and research purposes. / 20
Additional products / Tumor necrosis factor, therapeutic enzymes / 14
The size of the biopharmaceutical market is sizeable. Some of the leading approved biopharmaceutical products are listed below.
Table 2.2 Approximate annual market values of approved biopharaceutical products.
Procrit (Amgen/Johnson & Johnson) / Erythropoietin (treatment of anemia) / 2.7
Epogen (Amgen) / Erythropoietin (treatment of anemia) / 2.0
Intron A (Schering Plough) / Interferon-a (treatment of leukemia) / 1.4
Neupogen (Amgen) / Colony stimulating factor (treatment of neutropenia) / 1.2
Avonex (Biogen) / Interferon-b (treatment of multiple sclerosis) / 0.8
Embrel (immunex) / Monoclonal antibody (treatment of rheumatoid arthritis) / 0.7
Betasteron (Chiron/Schering Plough) / Interferon-b (treatment of multiple sclerosis) / 0.6
Cerezyme (Genzyme) / Glucocerebrosidase (treatment of Gaucher’s disease) / 0.5
At the low-dollar end, proteins are produced in bulk quantities for the food, chemical, and pharmaceutical industries. Unlike the biopharmaceutical proteins, these industrial enzymes do not require rigorous purification and can be produced in larger and less expensive processes. Bulk enzymes have a billion-dollar annual market, by far due to proteases used in detergents. Some examples of these types of protein products are listed below.
Table 2.3. Some enzyme products for industrial applications
Enzyme / Industrial applicationProteases / Inclusion in detergent preparations
Cheese- making
Brewing/baking industries
Meat/leather industries
Animal/human digestive aids
Amylases / Starch processing industries
Fermentation/ethanol production industries
Cellulases/hemicellulases / Brewing industry
Fruit juice production
Animal feed industry
Pectinases / Fruit juice/fruit processing industry
Glucose isomerase / Production of high-fructose syrups
Lipases / Dairy industry
Vegetable oil industry
Chemical industry
Cyclodextrin glycosyltransferase / Productions of cyclodextrins for the pharmaceutical and other industries
Penicillin acylase / Production of semisynthetic penicillins
Sources of Protein Products
While bulk enzymes produced for the food and chemical industries are most often isolated directly from microbial or plant sources, biopharmaceuticals are more often isolated from recombinant organisms. Although biopharmaceutical protein products such as insulin were originally isolated from human and animal tissues, they are not likely to found in these natural sources in high concentrations, making the extraction and purification of these proteins prohibitively expensive. Also, contaminating residuals from natural sources can be unsafe, whether due to allergic responses in patients or due to contaminating viruses or prions.
These disadvantages can be overcome by using a recombinant productions system. By isolating the gene coding for a specific protein and cloning it into a high-expression vector in a recombinant host, the possibility of contaminating viruses and prions can be eliminated. The higher level of expression of the protein in a recombinant host can greatly reduce purification costs, and protein engineering can be used to design improvements in stability or effectiveness of a protein product.
The table below lists some expression levels of biopharmaceutical proteins in a bacterial expression host, Escherichia coli. While these proteins isolated from human and animal tissue sources might be at levels nearly indetectable, they can become the dominant protein expressed in a recombinant organism.
Table 2.4. Heterologous protein expression in E. coli
Protein / Expression level(% of total protein)
Insulin / 20
Bovine growth hormone / 5
Interleukin 2 / 10
Human tumor necrosis factor / 15
interferon g / 25
E. coli was the first host used for production of recombinant proteins because it was well understood genetically and was very amenable to transformation and expression of recombinant genes. Its fermentation characteristics were also well understood. Not all proteins are expressed well in E. coli, however, in part due to the bacterial host’s inability to perform necessary post-translational modifications to recombinant proteins. Also, E. coli produces an endotoxin that acts as a pyrogen when injected, and this endotoxin is very difficult to purify from an E. coli fermentation.
In more recent years, however, the biotechnology industry has turned to alternative hosts for recombinant hosts in productions systems: fungal, plant, and animal tissue culture. Below is a table outlining some examples of production hosts for recombinant proteins, giving some examples of recombinant therapeutic proteins approved for general medical use that are produce in them, along with some advantages that these hosts present.
Table 2.5. Recombinant hosts used for protein production
Recombinant host / Some approved therapeutic proteins in production / Advantages of hostSaccharomyces cerevisiae (yeast) / Novolog (engineered insulin)
Leukine (colony stimulating
factor)
Recombinvax, Comvax, Infanrix, Twinrix, Primavex, Hexavax (subunit vaccines)
Regranex (platelet-derived
growth factor) / Well characterized genetics & fermentation
GRAS (“generally regarded as safe”) by regulators
Rapid and inexpensive fermentations
Can carry out some post-translational
modifications of proteins
Insect cells / Bayovac CSF E2 and Porcilis Pesti (swine flu subunit vaccines) / High-level recombinant protein expression
Performs post-translational modifications
Can be engineered to secrete recombinant
Proteins
Human pathogen-free
Cheaper to culture than mammalian cells
Mammalian cells / Insulins
Tissue plasminogen activator
Follicle-stimulating hormone
Interferon-b
Erythropoietin
Glucocerebrosidease
Factor VIIa
Vaccines / Ability to carry out necessary post-
translational modifications
protein glycosylation patterns most closely
mirrors that found in humans
Downstream Processing of a Protein Product
There is no single best way to purify a given protein. The optimal protein purification strategy depends on the properties of the protein being purified, the starting concentration of the protein being purified, and the types of contaminating materials that it is being purified from. Most proteins produced commercially rely on fermentation by microbial or animal cell culture. The process of harvesting and purifying a protein being produced in an industrial setting is referred as “downstream processing.” It includes all steps of production downstream of the fermentation step. Since downstream processing of a protein can often exceed all other costs of production combined, it must be a carefully designed strategy, often requiring extensive development by scientists and engineers. An optimal purification scheme results in a maximal yield with the fewest and least expensive of steps of purification.
The following outlines the general steps that are part of downstream processing of proteins. Each step will be discussed in greater detail later in this lab manual.
1. Since most proteins are not secreted from cells, the first step of downstream processing generally consists of a cell disruption step, followed by removal of unbroken cells and cell debris. Cell disruption can be done by a relatively mild treatment with chemicals, or a more rigorous physical disruption by sonication or homogenization. Clearing the lysate of insoluble debris can be done by centrifugation or by filtration. Partitioning between two immiscible liquid phases can also be used in some cases.
2. Since processing of large volumes is expensive, the first purification step usually includes concentrating the protein extract to a smaller volume. This can be done by precipitating the protein, by adsorbing the protein to a column such as ion exchange, or by ultrafiltration through a membrane of a pore size that does not allow the protein to pass through.
3. Once the protein solution volume has been reduced to a more manageable size, purification can proceed by a number of techniques. Chromatography offers the highest resolution, but generally speaking one chromatographic step is not sufficient to purify the protein to homogeneity. Some types of chromatography that can be used include:
a. size exclusion chromatography (molecular sieving)
b. ion-exchange chromatography
c. hydrophobic interaction chromatography
d. affinity chromatography
e. adsorption chromatography on hydroxyapatite
4. For biopharmaceutical products that require higher levels of purity and can command a higher price in the marketplace, some more sophisticated techniques can be used to purify a protein. These include immunoaffinity techniques and high performance liquid chromatography (HPLC).
5. When the protein has been purified sufficiently, it is either dried by lyophilization or freeze-drying techniques, or it is formulated into a solution that stabilizes its activity and integrity.
Green Fluorescent Protein
In this lab module, we will purify the green fluorescent protein (GFP), a fluorescent protein naturally occurring in the Pacific jellyfish Aequoria victoria that has been successfully cloned into a number of organisms from bacteria to mice. Although originally chosen for its novelty of causing the transgenic organisms to glow green, GFP has been successfully used as a marker for transformation. Recent studies have created gene fusion in which the GFP gene is fused to genes of target markers on either the N- or C-terminus of the protein that they encode. The GFP becomes a marker for the intracellular location of the target gene product, tracking its migration by fluorescence microscopy into the nucleus, mitochondria, secretory pathway, plasma membrane or cytoskeleton. GFP can also be used as a reporter of gene expression levels as well as a measure of protein-protein interactions. Therefore, GFP is a very useful tool for both geneticists and for cell biologists.
The green fluorescent protein is a medium-sized protein of 238 amino acids and a molar mass of 27,000 daltons. In spectrophotometry it shows a major absorption peak at 395 nm and a minor absorption peak at 475 nm. The characterizing molar extinction coefficients are 30,000 and 7,000 M-1cm-1 respectfully. Fluorescence at 508 nm is not energy requiring and depends on the amino acids serine-65, tyrosine-77, and glycine-67. This trimer forms a fluorescent chromophore after translation by cyclization and oxidation reactions.
Once isolated, the GFP is stable across a wide range of temperatures and pH. It is very resistant to denaturation, requiring treatment with 6 M guanidine hydrochloride at 90oC or pH of <4.0 and >12.0. Furthermore, it is able to renature completely within minutes following many denaturing protocols, including sulfhydryl reagents such as 2-mercaptoethanol.
GFP consists of a dimer, each made of a barrel-shaped cylinder made primarily of b pleated sheets on the outside and a-helices on the inside, a structure that is unique among proteins. This structure produces a compact domain that surrounds and protects the fluorophore located at the center of each cylinder as shown in Fig. 2.1. The N-terminal region of the protein acts as a “cap” on the end of the protein, further protecting the core fluorophore. When this cap is disrupted, the fluorescence may be easily quenched. The dimers are probably held together with the hydrophilic interactions of the pleated sheets on the outside of the cylinders.
Fig 2.1: Overall Shape of GFP Monomer (from Carson, M, 1987. J. Mol. Graphics 5:103-106.)
In this module, we will extract GFP from transformed yeast cells by sonication, three-phase extraction, and homogenization by glass beads. In a later lab exercise, we will concentrate the GFP by precipitating it with ammonium sulfate and purify it by column chromatography. We will then check the purity of this isolated GFP by SDS-PAGE electrophoresis.
These techniques of cell disruption, protein extraction, protein precipitation, column chromatography and electrophoresis are basic techniques used in labs for isolating and characterizing many different types of proteins including enzymes. We will be using GFP as the protein of choice because it glows green under UV light and therefore readily visualized.
Lab 2-A:
Preparation of Reagents
Introduction:
The ability to make reagents is an essential skill for any biotechnicians. The accuracy of calculation and of measurement is critical to the outcome of any experiment, whether it be one you do yourself or one in which you prep for someone else. There are several critical aspects to making solutions that should be followed at all times.
Ø Check and recheck each calculation. It is best if two people make a calculation independently and then cross check their answers.
Ø Read each reagent bottle twice, once before using and once afterwards. This helps ensure that the right reagent is used.
Ø Complete a media prep form for every solution you prepare. This should include the formula, with the supplier and catalog number if available as well as the concentration and the amount weighed out for each reagent. Some media prep forms will also have space to include the balance number, pH meter number and other pieces of important information.
Ø Label each bottle before filling. Write down the name of the solution, your initials and the date. Some industries have special blank labels to be used for each reagent. Others use tape and a permanent marker.