The Origins of Recombinant Proteins

Molecular biology is the fascinating field of biology that studies macromolecular mechanisms within our cells, combining aspects from several branches of science. Today, we have a vast array of laboratory techniques used to further our understanding of molecular biology. These techniques have evolved over the years with advancements in each field to establish what are now routine procedures, but this didn’t happen overnight! Here, some of the pioneering studies in molecular biology that have subsequently allowed Peak Proteins, and many others to study recombinant proteins are explored.

Some ground breaking discoveries in the first half of the 21^st century lead to a “golden age of molecular biology”. Historic discoveries from this time included Avery’s conclusion that genetic material is formed of DNA, Beadle and Tatum’s one gene-one enzyme rule and the pinnacle of Watson and Crick solving the structure of the DNA double helix using X-ray fibre diffraction and by bringing together the separate findings of other pioneers, such as Rosalind Franklin. This new information opened the door for the next phase of discovery, one in which others would take these findings and develop the processes that we use in the lab today. The first breakthrough came in understanding the central dogma of biology, how DNA makes RNA which makes protein. The 1968 Nobel Prize in Physiology or medicine acknowledged the work conducted by Khorana, Nirenberg and Holley in deciphering the triplet nature of the genetic code, and its function within protein synthesis. The first codon determined by Nirenberg was found by supplying an individual bacterial cell with radioactive amino acids. Analysis later showed the only synthesised protein to retain radioactivity was that containing the supplied phenylalanine (UUU). For the first time, this provided a real understanding of how proteins are formed at the molecular level and offered an opportunity to one day manipulate the genetic make-up of living organisms.

Resolving the alphabet from which the language of proteins is written greatly helped progress to the next step: isolating a single gene. The Lac operon found in E. coli was already known for its role played in lactose metabolism and helped in the discovery that proteins are only expressed when they are needed. One component of the Lac operon, lacZ, was the first gene isolated in 1965. The gene, lacZ, encodes for b-galactosidase, an enzyme used to digest lactose, a sugar found in milk. James Shapiro and Jon Beckwith utilised methods involving viral vectors that when combined formed double stranded lacZ DNA, meaning the single stranded viral DNA could be degraded using a single-strand specific DNase. Isolating the first gene opened the door to future groups studying the impact that single genes can have on many biochemical systems.

The next leap in the story of recombinant protein production came in the isolation two essential enzymes: DNA ligase and restriction enzymes. DNA ligase was first characterised in 1967 following an arduous 6-step purification by Gellert, Lehman, Richardson and Hurwitz and later supplied to early researchers following purification from bacteria found in the environment! Fortunately for us at Peak Proteins, the complex and time-consuming techniques used in the early purifications of DNA ligase are rarely used today, thanks to future breakthroughs in this field. DNA ligase catalyses the formation of a phosphodiester bond, the backbone of DNA and RNA strands and because of this, it is often described as molecular glue.

In contrast, restriction enzymes have the ability to cleave DNA molecules into fragments. Currently, four groups of naturally occurring restriction enzymes (types I-IV) exist and they are categorised based on specific sites on the DNA they cleave. Together, both enzymes can be used in tandem to manipulate DNA; removing and adding sequences of interest into specific vectors and leading to the development of hybrid DNA, commonly known as recombinant DNA.

The general definition of recombinant DNA is the addition of two genetic fragments from distinct sources, such as adding human DNA to bacterial DNA. With the generation of single genes and means of cutting and joining DNA, scientists of the time were now able to clone genes into other systems so that human genes could be placed into bacterial plasmids. Paul Berg, the winner of the 1980 Nobel Prize in Chemistry, was the first to combine the information known at the time to create the first recombinant DNA. Berg was first able to cut DNA from the bacterial lambda virus using a purified restriction enzyme, EcoRI, and incorporate this fragment into the genetic material of the simian virus, SV40, using a DNA ligase.

Although the modern day uses of this technique remained unknown at the time, the potential this technique held were certainly understood. Stanley Cohen and Herbert Boyer quickly utilised the techniques pioneered by Berg to develop the first recombinant organism. Together, they were able combine Cohen’s findings on manipulating bacterial plasmids by stimulating their removal and return to bacterial cells and Boyer’s findings that restriction enzymes did not cleave DNA cleanly, leaving sticky ends that could be taken advantage of to transfer the gene for frog ribosomal RNA into E. coli. Thanks to this, they were able to trigger the field of scientific cloning, enabling scientists to clone genes of interest into plasmid vectors. Today, Peak Proteins use this technology to add genes of interest into vectors that contain affinity tags, facilitating our purification methods. If you are interested in reading about protein tags and their applications, read our blog that discusses the commonly used protein tags.

Historically, large-scale protein production required harvest and isolation from a native plant or animal source. Thanks mainly to the work described above, we now rely on recombinant protein technology, along of course with bacterial, yeast, insect and mammalian expression hosts, to efficiently produce and isolate proteins that contribute to medicinal, agricultural and industrial advancements every day. If you need help designing, expressing or purifying a recombinant protein of interest, get in touch via our web site or info@peakprotein,.com to see how we can help.

Written by Scott Allen

References:

Avery, O., MacLeod, C. and McCarty, M., 1944. STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES. Journal of Experimental Medicine, 79(2), pp.137-158. (10.1084/jem.79.2.137)

Beadle, G. and Tatum, E., 1941. Genetic Control of Biochemical Reactions in Neurospora. Proceedings of the National Academy of Sciences, 27(11), pp.499-506. (10.1073/pnas.27.11.499)

WATSON, J. and CRICK, F., 1953. Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. Nature, 171(4356), pp.737-738. (10.1038/171737a0)

Matthaei, J., Jones, O., Martin, R. and Nirenberg, M., 1962. CHARACTERISTICS AND COMPOSITION OF RNA CODING UNITS. Proceedings of the National Academy of Sciences, 48(4), pp.666-677. (10.1073/pnas.48.4.666)

Nirenberg, M., Leder, P., Bernfield, M., Brimacombe, R., Trupin, J., Rottman, F. and O’Neal, C., 1965. RNA codewords and protein synthesis, VII. On the general nature of the RNA code. Proceedings of the National Academy of Sciences, 53(5), pp.1161-1168.

SHAPIRO, J., MACHATTIE, L., ERON, L., IHLER, G., IPPEN, K. and BECKWITH, J., 1969. Isolation of Pure lac Operon DNA*. Nature, 224(5221), pp.768-774. (10.1038/224768a0)

Shuman, S., 2009. DNA Ligases: Progress and Prospects. Journal of Biological Chemistry, 284(26), pp.17365-17369. (10.1074/jbc.R900017200)

Jackson, D., Symons, R. and Berg, P., 1972. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli. Proceedings of the National Academy of Sciences, 69(10), pp.2904-2909. (10.1073/pnas.69.10.2904)

Sanger, F., Nicklen, S. and Coulson, A., 1977. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, 74(12), pp.5463-5467 (10.1073/pnas.74.12.5463)

Jackson, D., Symons, R. and Berg, P., 1972. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli. Proceedings of the National Academy of Sciences, 69(10), pp.2904-2909. (10.1073/pnas.69.10.2904)