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Challenges and solutions for recombinant proteins production

Challenges and solutions for recombinant proteins production

A pill of history

That bacteria can acquire DNA from outside has been known since 1928 following Griffith’s experiment. However, it was necessary to wait until the second half of the last century for recombinant DNA technology to develop. The discovery of plasmids in the 1950s, restriction enzymes and DNA ligases in the 1960s signified the foundations for the development of recombinant DNA technology, which has profoundly revolutionised biomedical sciences with countless applications in basic and industrial research and drug development.

Essential elements in the recombinant DNA technology

  • Plasmids are circular DNA molecules capable of replicating autonomously in bacterial cells to which they can confer particular features, such as resistance to an antibiotic or the ability to metabolise a substance. Plasmid
  • Restriction enzymes are endonucleases capable of cutting DNA at specific sites, recognising a specific sequence (base combination) and generating ends in the DNA molecule that can serve as attachment sites for DNA fragments with compatible ends. Restriction enzymes
  • DNA ligases are enzymes capable of reforming the phosphodiester bond between compatible DNA ends, restoring the original DNA structure or enabling the insertion of a new DNA fragment (cloning). Dna ligase

Transformation is the process through which a recombinant plasmid produced by cutting and pasting the foreign DNA is introduced in a host cell capable of replicating the plasmid or even expressing the foreign DNA. Dna transformation

In the 1970s, additional fuel in the development of recombinant DNA technology was provided through the discovery of the enzyme retro-transcriptase, which allows the obtaining of a DNA molecule suitable for cloning starting from the RNA transcript.

Another disruptive discovery was the PCR (polymerase chain reaction) in 1983, which allows the amplification of a rare or diluted DNA fragment from a heterogeneous mixture, resulting in enough DNA for subsequent manipulations.

In the timeline above reported, there are the indications of the first human recombinant proteins produced, insulin (1978) and growth hormone (1981) as well as the largest one produced for the first time in the early 1990s, the coagulation factor VIII. While insulin and growth hormone are small proteins, the coagulation factor VIII is a very large protein, comprising a number of post-translational modifications (PTMs).

What is needed to produce a recombinant protein

We have already understood that to produce a recombinant protein, we need a plasmid, i.e. the vector delivering the DNA sequence coding for the protein of interest (POI) inside the host, restriction enzymes, ligases, and host cells, such as E. coli. However, another essential element must be present in the plasmid for the synthesis of the protein, namely the promoter.

Recombinand protein process

The promoter is a DNA sequence, located upstream of the DNA fragment coding for POI, which allows the RNA polymerase of the host cells to bind the plasmid at the right position to start transcription (i.e. the synthesis of the mRNA). After transcription, mRNA is translated into proteins by ribosomes, t-RNAs, and any other factors normally used by the host cell to synthesize its own proteins. The last step is the folding process, which normally need the help of chaperone proteins, to eventually reach the native 3D structure.

Some proteins need additional steps of maturation to become fully functional, globally named post translational modification (PTM), and comprising either glycosylation, phosphorylation, acetylation, proline isomerization etc. PTM

The production of a recombinant protein requires careful and accurate design of the process, involving the choice of the expression system and consequently the host organism as well as the identification of the best purification method so as to optimize yield, time and cost of production, while ensuring the final quality of the protein of interest.

Furthermore, specific characteristics of the POI such as the presence of hydrophobic stretches of amino acids, disordered regions, quaternary structure, cofactors or prosthetic groups can affect the final result. Additional features that can be added to the protein are sequences, namely tags, useful for the process of protein purification by affinity chromatography towards the tag. Protein purification

Tags may also help folding, stability and solubility of the fusion protein. In case the POI needs to be free of any additions, the tag can be removed simply by inserting a site for a specific protease that after purification can be added to detach the tag. Different types of tags can be fused to the POI either at the N-ter or C-ter and cognate binders can be used for the purification. A few examples are reported in the table.

Tag Sequence Binder
Poly-his HHHHHH Nickel-beads
HA YPYDVPDYA Antibody anti-HA
Flag DYKDDDDK Antibody anti-Flag
Glutathione-S-transferase (GST) Sequence of 211 aa, 26 kDa Glutathione
Maltose Binding Protein (MBP) Sequence of 370 aa, 42.5 kDa Amylose

Different expression systems according to the POI

Depending on the type of protein, it is possible to chose among different expression systems and host cells. This is much related to the dimension of the POI and the type of PTMs that must be introduced to obtain a functional protein. tab1 tab2 tab3 tab4

Recombinant protein production issues / possible reasons / conceivable solutions

Low or no yield can be achieved in the production of recombinant proteins even though a careful design has been done. The reasons for such a result can be sought at various levels:

Issue Possible reasons Conceivable solutions
Low transcription Promoter may not be suitable Change promoter; try an inducible promoter
Low protein translation Check ribosome binding site (RBS); check codon usage Mutagenize the cDNA to introduce suitable RBS or codons for specific amino acid
Low protein yield Proteases might degrade the POI; check for protease site in the protein sequence Change bacterial strain; mutagenize the POI sequence to remove the protease site; try protein secretion; fuse protein to a host protein/tag (chimeric protein)
High yield but protein aggregates The folding system is not appropriate; check POI for transmembrane (TM) domains, exposed hydrophobic regions, and disordered regions Use inducible promoters, try different expression systems, try to overexpress protein chaperons; see if it is possible to delete problematic regions of the POI; use detergent during purification steps
Poor recovery of the purified protein No suitable system for direct purification Fuse POI to a tag; fuse POI to a host protein (chimeric protein); change lysis method

As evidence of the enormous impact that this area of research and of industrial production has and will have in the near future, here is a graph showing the trend in the size of the world market for recombinant proteins.

© University of Padova
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