Master the Steps of Recombinant Protein Purification

DNA sequencing

Type I

Electrophoresis

Herbert Boyer

Once every 4096 base pairs

Once every 256 base pairs

GST Bind™ Purification Kits

Its unique identifying property

The reaction rate

To recognize the protein target

High yields

Yeast, Virus, Plants

Pichia pastoris

Pichia pastoris

S. cerevisiae

E. coli system

Mammalian cell line expression

Mammalian cell line expression

Species identification of ocular isolates of methicillin resistant staphylococci

To carry up to 20 kb of foreign DNA

Origin of replication

To cut DNA segments using specific restriction enzymes

Transformation

Natural sources are often rare and expensive

Design expression plasmid, transform, select

Eukaryotic vector

To facilitate protein purification

Affinity chromatography

To mimic the effect of lactose

To encode an affinity tag

Restriction enzymes, electrophoresis, polymerase chain reaction (PCR)

To create a fingerprint of a DNA molecule based on the pattern of DNA fragments produced by a restriction enzyme

once/4096 bp

Berg, Boyer, and Cohen

The histidine tag is used for affinity purification of the protein by binding to certain metals such as nickel.

Some problems with immunoaffinity chromatography include the high cost and difficulty in purifying monoclonal antibodies, the potential for other proteins to bind non-specifically or inactivate the antibodies, and the possibility of the antibodies leaching off the column during purification.

Affinity columns are typically used as a last resort in the purification process, when the volume has been reduced and the majority of contaminants have already been removed. They are used to specifically bind and isolate the target protein.

Advantages of using bacterial systems include quick growth, high yields, low cost of media, and low fermentor costs. However, bacterial systems have difficulty expressing large proteins, lack glycosylation and signal peptide removal capabilities, and may not be suitable for S-S rich proteins.

Polylinkers, also known as multiple cloning sites, are inserted DNA fragments with multiple recognition sequences for restriction enzymes. They allow for the efficient and precise insertion of target DNA segments into a vector by cutting the target DNA segment using a specific restriction enzyme and then inserting it into the vector.

The essential elements of a plasmid vector include:1. Origin of replication: allows the plasmid to replicate independently within a host cell.2. Antibiotic resistance gene: provides a selective advantage to cells containing the plasmid.3. Multiple cloning site: a region of the plasmid that contains multiple recognition sequences for restriction enzymes, allowing for the insertion of target DNA segments.4. Promoter: initiates transcription of the target gene.5. Terminator: signals the end of transcription.6. Selectable marker: allows for the identification and selection of host cells that have taken up the plasmid.

The process of introducing DNA into bacterial cells is called transformation.

The purpose of recombinant protein expression is to produce large amounts of a specific protein of interest. This is usually done using recombinant DNA technology, where the gene encoding the protein is inserted into an expression vector and then introduced into a host organism, such as bacteria or yeast. Recombinant protein expression allows for the production of proteins that are difficult to obtain from natural sources, can be modified for specific applications, and can be produced in large quantities.

The steps involved in recombinant protein purification are: 1. Design expression plasmid, transform, select. 2. Grow culture of positive clone, induce expression. 3. Lyse cells. 4. Centrifuge to isolate protein-containing fraction. 5. Column Chromatography—collect fractions. 6. Assess purity on SDS-PAGE.

The requirements for vector selection in recombinant protein expression include: 1. Compatibility with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells). 2. Inducible strong promoters. 3. Ribosome binding sites. 4. Termination sequences. 5. Affinity tag or solubilization sequences. 6. Polylinker site.

Some strategies for engineering proteins for ease of purification and detection include: 1. Adding a signal sequence that causes secretion into culture medium. 2. Adding a protein that helps the protein refold and stay soluble. 3. Adding a sequence that aids in precipitation. 4. Adding an affinity handle (such as a His-tag). 5. Adding a sequence that aids in detection.

The principle behind affinity chromatography for protein purification is the specific binding interaction between a target protein and an immobilized ligand on a chromatography column. The target protein binds to the ligand while non-specifically or weakly bound proteins are washed off. The target protein can then be eluted using a competitor or a protease.

Advantages of using yeast as an expression system for protein production include quick growth, high yields, low cost of media, ability to express large proteins, glycosylation and signal peptide removal, and chaperonins to help fold 'tough' proteins. Disadvantages include the need for specific yeast strains, the time-consuming process of set-up, and the limited sustainability of cell culture.

Advantages of using baculovirus as an expression system for protein production include the ability to express large proteins, correct glycosylation and signal peptide removal, and the presence of chaperonins to help fold 'tough' proteins. Disadvantages include slow growth, the need for specific insect cells, and the time-consuming and complex set-up process.

Advantages of using mammalian cells as an expression system for protein production include the ability to express large proteins, correct glycosylation and signal peptide removal, and the presence of chaperonins to help fold 'tough' proteins. Disadvantages include the time-consuming and costly set-up process, limited sustainability of cell culture, and modest yields.

The choice of expression system for protein production depends on factors such as the size and characteristics of the protein. For large proteins, eukaryotic systems are preferred. Small proteins can be expressed in either prokaryotic or eukaryotic systems. Other factors to consider include the need for correct glycosylation and signal peptide removal, the folding assistance provided by chaperonins, and the scalability and cost of the expression system.