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Overview of Protein Expression

Protein expression refers to the way in which proteins are synthesized, modified and regulated in living organisms. In protein research, the term can apply to either the object of study or the laboratory techniques required to manufacture proteins. This article focuses on the latter meaning of protein expression. However, in practical terms, recombinant protein production depends on using cellular machinery.

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Introduction to Protein Expression

Proteins are synthesized and regulated depending upon the functional need in the cell. The blueprints for proteins are stored in DNA and decoded by highly regulated transcriptional processes to produce messenger RNA (mRNA). The message coded by an mRNA is then translated into a protein. Transcription is the transfer of information from DNA to mRNA, and translation is the synthesis of protein based on a sequence specified by mRNA.

Diagram of transcription and translation
Simple diagram of transcription and translation. This describes the general flow of information from DNA base-pair sequence (gene) to amino acid polypeptide sequence (protein).

In prokaryotes, the process 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, or protein synthesis, occurring in the cytoplasm.

 

Prokaryotic vs. eukaryotic transcription and translation
Comparison of transcription and translation in prokaryotes vs. eukaryotes.

Transcription

Transcription occurs in three steps in both prokaryotes and eukaryotes: initiation, elongation and termination. Transcription begins when the double-stranded DNA is unwound to allow RNA polymerase binding. Once transcription is initiated, RNA polymerase is released from the DNA. Transcription is regulated at various levels by activators and repressors and also by chromatin structure in eukaryotes. In prokaryotes, no special modification of mRNA is required and translation of the message starts even before the transcription is complete. In eukaryotes, however, mRNA is further processed to remove introns (splicing), addition of a cap at the 5´ end and multiple adenines at the mRNA 3´ end to generate a polyA tail. The modified mRNA is then exported to the cytoplasm where it is translated.

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In vitro transcription methods

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Translation

Translation or protein synthesis is a multi-step process with initiation, elongation and termination steps. The process requires macromolecules like ribosomes, transfer RNAs (tRNA), mRNA, and protein factors as well as small molecules like amino acids, ATP, GTP, and other cofactors. There are specific protein factors for each step of translation (see table below). The overall process is similar in both prokaryotes and eukaryotes, although particular differences exist.

During initiation, the small subunit of the ribosome bound to initiator t-RNA scans the mRNA starting at the 5’end to identify and bind the initiation codon (AUG). The large subunit of the ribosome joins the small ribosomal subunit to generate the initiation complex at the initiation codon. Protein factors as well as sequences in mRNA are involved in the recognition of the initiation codon and formation of the initiation complex. During elongation, tRNAs bind to their designated amino acids (known as tRNA charging) and shuttle them to the ribosome where they are polymerized to form a peptide. The sequence of amino acids added to the growing peptide is dependent on the mRNA sequence of the transcript. Finally, the nascent polypeptide is released in the termination step when the ribosome reaches the termination codon. At this point, the ribosome is released from the mRNA and is ready to initiate another round of translation.

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In vitro translation methods

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Protein synthesis machinery. Summary of the primary components and features of prokaryotic and eukaryotic translational apparatus.
Component Prokaryotes Eukaryotes
Ribosomes 30S and 50S Subunits 40S and 60S Subunits
Template or mRNA

No further processing of mRNA transcript occurs after transcription.

mRNA is polycistronic and contains multiple initiation sites.

After transcription, the mRNA transcript is spliced to remove the non-coding regions (introns), and a cap structure (M7methyl gaunosine) and a poly adenosine sequence are added at the 5' and 3' end of the message respectively.

The Cap structure and the poly A are important for export of mRNA to the cytoplasm, proper initiation of translation and stability of mRNA among other functions. The mRNA is usually monocistronic.

Features of translation

The Shine-Dalgarno sequence is present on the mRNA transcript, and a complementary sequence is present in the ribosomal subunit. This facilitates binding and alignment of the ribosome on the mRNA at the translation initiation site (AUG).

The first amino acid of the nascent polypeptide is formylated methionine.

Translation initiation occurs in two ways:

Cap-dependent translation: Cap structure and the cap binding proteins are responsible for proper ribosome binding to mRNA and recognition of the correct initiation codon. The first AUG codon in the 5’end of mRNA functions as the initiation codon. Sometimes Kozak sequence may be present around the initiation codon.

Cap-independent translation: Ribosome binding to mRNA occurs through 'internal ribosome entry site' (IRES) on mRNA.

Initiation factors Three initiation factors are known, IF1, IF2, &IF3 More than three Initiation factors, which are regulated by phosphorylation. The Initiation step is the rate-limiting step in eukaryotic translation.
Elongation factors EF-Tu & EF-Ts, EF-G EF1(α, β, γ) and EF2
Termination or release factors RF1 and RF-2 eRF-1

Post-Translational Modification

After translation, polypeptides are modified in various ways to complete their structure, designate their location or regulate their activity within the cell. Post-translational modifications (PTMs) are various additions or alterations to the chemical structure and are critical features of the overall cell biology.

Types of post-translational modification include:

  • Polypeptide folding into a globular protein with the help of chaperone proteins to arrive at the lowest energy state
  • Modifications of the amino acids present, such as removal of the first methionine residue
  • Disulfide bridge formation or reduction
  • Protein modifications that facilitate binding functions:
    • Glycosylation
    • Prenylation of proteins for membrane localization
    • Acetylation of histones to modify DNA-histone interactions
  • Addition of functional groups that regulate protein activity:
    • Phosphorylation
    • Nitrosylation
    • GTP binding

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Overview of post-translational modification (PTM)

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Affinity Purification Kits
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Recombinant Protein Expression Methods

 

In general, proteomics research involves investigating any aspect of a protein such as structure, function, modifications, localization, or protein interactions. To investigate how particular proteins regulate biology, researchers usually require a means of producing (manufacturing) functional proteins of interest.

Given the size and complexity of proteins, chemical synthesis is not a viable option for this endeavor. Instead, living cells and their cellular machinery are usually harnessed as factories to build and construct proteins based on supplied genetic templates.

Unlike proteins, DNA is simple to construct synthetically or in vitro using well established recombinant DNA techniques. Therefore, DNA templates of specific genes, with or without add-on reporter or affinity tag sequences, can be constructed as templates for protein expression. Proteins produced from such DNA templates are called recombinant proteins.

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Recombinant Proteins

In vivo Protein Expression

Traditional strategies for recombinant protein expression involve transfecting cells with a DNA vector that contains the template and then culturing the cells so that they transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic in vivo protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield.

Bacterial protein expression systems are popular because bacteria are easy to culture, grow fast and produce high yields of recombinant protein. However, multi-domain eukaryotic proteins expressed in bacteria often are non-functional because the cells are not equipped to accomplish the required post-translational modifications or molecular folding. Also, many proteins become insoluble as inclusion bodies that are very difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding procedures.

Mammalian in vivo expression systems usually produce functional protein, but the yield is low, cost of production is high and mammalian cell culturing is time-consuming. In addition, in vivo systems are not conducive to either high throughput protein synthesis or expression of proteins that are toxic to host cells.

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Overview of protein extraction

 

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Inclusion Body
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In vitro (Cell-free) Protein Expression

Cell-free protein expression is the in vitro synthesis of protein using translation-compatible extracts of whole cells. In principle, whole cell extracts contain all the macromolecules components needed for transcription, translation and even post-translational modification. These components include RNA polymerase, regulatory protein factors, transcription factors, ribosomes, and tRNA. When supplemented with cofactors, nucleotides and the specific gene template, these extracts can synthesize proteins of interest in a few hours.

Although not sustainable for large scale production, cell-free protein expression systems have several advantages over traditional in vivo systems. Cell-free expression allows for fast synthesis of recombinant proteins without the hassle of cell culture. Cell-free systems enable protein labeling with modified amino acids, as well as expression of proteins that undergo rapid proteolytic degradation by intracellular proteases. Also, with the cell-free method, it is simpler to express many different proteins simultaneously (e.g, testing protein mutations by expression on a small scale from many different recombinant DNA templates).

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In vitro recombinant protein expression methods

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Thermo Scientific Human
In Vitro Translation Systems

Chemical Protein Synthesis

Chemical synthesis of proteins can be used for applications requiring proteins labeled with unnatural amino acids, proteins labeled at specific sites or proteins that are toxic to biological expression systems. Chemical synthesis produces highly pure protein but works well only for small proteins and peptides. Yield is often quite low with chemical synthesis, and the method is prohibitively expensive for longer polypeptides.

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Protein crosslinking and
chemical modification

Written and/or reviewed by Douglas Hayworth, Ph.D.

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