Recombinant Protein Expression

What is Protein Expression

Cells, the basic unit of life, must be able to make proteins using information encoded in its DNA as proteins are essential for cellular architecture, giving the cell a particular shape and structure. Based on the central dogma, genetic information flows from DNA to RNA to protein when DNA is transcribed into RNA, which in turn is translated into a protein.

Protein Expression Definition

Protein expression involves making an RNA copy of the DNA code, a process called Transcription and translating information carried by nucleic acids to give the sequences of amino acids that make up proteins. The latter process is called Translation. In other words, the protein expression process includes gene expression and protein synthesis steps.

Transcription
Transcription
DNA mRNA Protein

Gene Expression from Gene to Protein

Gene expression process in prokaryotes is different from those of eukaryotic proteins.

In prokaryotes, the processes of transcription and translation occur simultaneously. The translation of mRNA starts even before a mature mRNA transcript is fully synthesized. This simultaneous transcription and translation of a gene is termed coupled transcription and translation.

In eukaryotes, the processes are spatially separated and occur sequentially, with transcription happening in the nucleus and translation occurring in the cytoplasm. After translation, polypeptides are modified in various ways to complete their structure, designate their location, or regulate their activity within the cell.

Protein Expression Process

Expression of Eukaryotic Gene in Bacteria

Although eukaryotic cells can be used to express eukaryotic proteins, bacteria are simpler to grow and manipulate genetically. Therefore, it is often desirable to express eukaryotic proteins in bacteria.

What are recombinant protein production steps?

Eukaryotic genes must be adapted for expression in bacteria. First, the mRNA from the gene of interest is converted to cDNA to provide uninterrupted coding DNA. The cDNA is cloned between a bacterial promoter and a bacterial terminator so the bacterial transcription and translation machinery express the coding sequence.

What is Protein Expression Vector?

In production of recombinant protein, the gene for the protein of interest is cloned into a vector and expressed into protein in a model organism.

The plasmids of the copy of the interest gene, or the expression vectors, are often used to enhance gene expression. These vectors provide a strong promoter to drive expression of the cloned gene. Expression vectors also contain genes for antibiotic resistance to allow selection of the vector and the recombinant protein.

Expression vectors are used to make eukaryotic proteins in bacteria. The vector has the ribosome-binding site, terminator sequences, and a strong regulated promoter. The eukaryotic gene is a cDNA copy of the mRNA. Once cloning is completed, plasmids are taken up into competent cells (chemically competent or electro competent E. coli) for propagation and storage, by a process called transformation.

Gene Expression Procedure

Because bacteria cannot process introns so it is standard procedure to clone the cDNA version of eukaryotic genes, which lacks the introns and consists solely of uninterrupted coding sequence. And the cDNA version of eukaryotic genes is generally used, even for expression in eukaryotic cells.

Protein Synthesis Procedure

In the translation step, adding some rare tRNA or changing the rare codon wobble position may help when the sequence for the recombinant protein encodes a rarely used tRNA, the protein production slows.

If recombinant proteins fold aberrantly, they aggregate and form inclusion bodies. If, during translation, molecular chaperone proteins hold the recombinant protein, the new polypeptide will fold properly and become fully functional. To enhance protein stability, the recombinant protein can be coexpressed with a molecular chaperone that helps the protein fold correctly. Alternatively, inclusion bodies can be purified and attempts made to refold the protein.

References

Assenberg R, et al. (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23(3): 393-402.
Rosano GL, et al. (2014) Recombinant protein expression in escherichia coli: Advances and challenges. Front Microbiol 5: 172.

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