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Modern Harvesting of Natural Biomolecules – Big is Beautiful

September 6, 2011

It was noted in the previous post that most natural molecules of use to humans (‘chrestomolecules ) have traditionally been of low molecular weight, with the exception of simple biopolymers. Here it is time to have a brief look at the scope of large molecules and even larger biological systems which can be sampled from the biosphere and usefully applied to human activities.

Big Uses for Big Molecules….

Large biomolecules such as proteins can obviously act as nutrients, but this special category of ‘usefulness’ is not the focus of interest here. Indeed, in this context it should be emphasized that the benefits of consuming a protein as a foodstuff for any organism (humans included) result from the acquisition of its amino acid building blocks. ‘Digestion’ is intimately associated with breaking down consumed nutrients into components which an organism can employ for its own biosynthetic requirements. The original three-dimensional structure and function of the consumed protein is clearly irrelevant for such a nutritive purpose. But when we are considering the utility of large biomolecules for other human needs, the protein shape and function is crucial.

Biotechnological developments in the past century or so have extended the field of molecular utility to include a diversity of proteins (noted in part of in the ‘chrestomolecule’ Table of a previous post). The great majority of these can be grouped into the functional categories of natural biocatalysts (enzymes) and natural specific binding molecules (antibodies), but some additional cases can be noted:

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Let’s take a quick look at some specific examples among these large protein sets:

Looking for Enzymes in All the Right Places

 Many enzymes from a variety of biological sources are used in industrial processes, and several examples are of particular interest from a molecular biological point of view. There is at this time considerable interest in biological resources obtainable from ‘extremophile’ micro-organisms, or literally ‘lovers of extreme environments’. Certain bacteria thrive under amazing extremes of temperature, salinity, pH or pressure, and bacterial biosystems mediating metabolic processes and growth must in consequence be selected for tolerance of each type of extreme conditions. Only in quite recent times has it become recognized that the earth harbors an entire thermoresistant prokaryotic ecosystem which lives by chemical energy independent of solar radiation. These organisms may be found in geothermal hot springs (as in Iceland or Yellowstone National Park), at deep underground sites or at thermal vents in the deep oceans.

Thermophilic (heat-stable) enzymes in general have many applications, but probably the most famous thermophile application in molecular biology is the use of the DNA polymerase from the hot-spring bacterium Thermus aquaticus, or Taq polymerase. (DNA polymerases are of fundamental importance for the copying and replicating of an organism’s DNA during cellular growth). The hardy Taq polymerase has made the polymerase chain reaction (PCR) a practical proposition, which has had an enormous impact in all areas of this and related sciences and gained its originator (Kary Mullis) a Nobel Prize. PCR involves replication and amplification of a DNA duplex in the laboratory through successive cycles of strand denaturation, association on each strand of specific primer oligonucleotides (short DNA segments), and primer extension (strand replication) with the DNA polymerase. Since strand denaturation is achieved by heating to 95˚ C or greater, a conventional polymerase present in the reaction is ‘killed’ and must be (very tediously) re-added for each cycle. A thermostable DNA polymerase, however, can be maintained throughout the course of the reaction. Although many other such heat-resistant polymerases are compatible with PCR, Taq polymerase is still the most widely used. At the other extreme from thermophilic organisms are psychrophiles, which thrive at very low polar temperatures, and are also potential sources of novel enzymes for novel applications.

 Antibodies

In the ‘chrestomolecule’ Table of a previous post, antibodies (immunoglobulins) were featured. These important effector molecules of the mammalian adaptive immune system can be regarded as natural binding molecules produced ‘to order’ in response to specific stimuli (usually threats from invading external organisms), by diversification and selection mechanisms. Their applications are many, but can be divided into in vivo tasks which relect the original biological role for antibodies, or in vitro roles. Even naturally-formed antibodies within an individual are obviously useful if they mediate recovery from microbial infections, and the production of antibodies within a host has long been artificially directed by means of vaccines. Relatively recently compared to the first use of vaccination (attributable to Edward Jenner and cowpox immunization against smallpox), passive immunity has emerged as a therapeutic tool. This can be effected by transfer of whole serum or serum components rich in immunoglobulins from immune animals or humans to non-immune recipients, for the purposes of their protection from sudden exposure to a toxic or infectious environmental agent. With passive immunization even with crude serum fractions, antibodies qualify as examples of large natural ‘chrestomolecules’. But in modern times, passive immunization goes far beyond this.

This essentially began with the discovery of monoclonal antibodies in 1975, and has led to the vast field of antibody engineering. The manipulation of antibodies has wide therapeutic applications, and products based on antibodies have become a billion-dollar earner for the biotechnology industry. But engineered (and conventional) antibodies have many uses in vitro as well, as noted above. These include diagnostics and sensors, and for a variety of purification purposes in research and industry. In its range, this topic really requires further attention in a later post. But before moving on, it’s worth noting that the fields of antibodies and enzymes are not mutually exclusive. For a couple of decades, it has been known that antibodies can be raised such that they catalyze specific chemical reactions. This is possible through clever immunization strategies, where host animals are immunized with analogs of chemical transition-state intermediates of enzymatic catalysis. Even so, the best artificially-generated catalytic antibodies fall short of the efficiencies observed with natural protein enzymes.

And Other Proteins….

Although antibodies remain the star players, the adaptive immune system can contribute other useful large molecules. The key adaptive receptor of the cellular arm of the vertebrate immune system is the T cell receptor, signaling through which activates various different classes of effector T cells. Engineered T cell receptors in a number of forms have application in some cutting-edge technologies for human therapies.  Some other proteins which modulate the functions of immune and other cells also have therapeutic and commercial significance. Included among these are interferons, known initially for their antiviral properties, but also for their immunomodulatory activities.

Beyond the immune system, various other proteins are mostly important through their contributions to knowledge, which have enabled the subsequent design of other useful structures. For example, the zinc-finger protein motif mediating DNA-binding has been intensively studied, allowing the derivation of a ‘code’ for binding and the generation of artificial binding motifs towards virtually any desired DNA sequence of 18 base pairs. When teamed with a nuclease domain, such tailored ‘zinc finger nucleases’ have tremendous potential in a number of clinical contexts. In different areas, numerous proteins have contributed their structural frameworks themselves for useful applications. This principle has been used for development of protein frameworks which can replace immunoglobulins for the specific recognition of desired binding targets.

….and even Bigger Higher-Level Useful Systems

By analogy with the introduced term ‘chrestomolecule’, an economically useful biological system (involving numerous mutually interactive molecules) could be termed a ‘chrestosystem’. But if we introduce biosystems into this picture, there are many levels of complexity that might apply. Does a whole organism thus qualify? Is even a domestic mammal a high-level ‘chrestosystem’? (After all, such animals have had many uses over the millennia in addition to being raised as food sources). And downward from there, certain fungi, yeast, and bacteria have long had uses in the modification of foodstuffs, long before they were even recognized as living organisms.

Perhaps cows (and so on) might indeed be viewed as simply highly complex organized systems in the service of humanity, but for the present purposes it will be most constructive to restrict our focus onto viruses. Viruses are usually considered as life-forms, and often have sophisticated regulatory controls, but are nevertheless many orders of magnitude less complex than a large multicellular organism. In their simplest forms, they can be regarded as small nucleic acid genomes packaged within a protein coat, and thus have a relatively small set of macromolecules which collectively allow them to infect their target cells and reproduce. So it is worth considering how some viral systems have useful properties for artificial exploitation. These will be grouped into bacterial viruses (bacteriophages, often referred to simply as ‘phage’) and a select group of animal viruses:

 The ‘other viral’ categories are relevant examples, and not intended to be exhaustively comprehensive. (Many other cases exist).

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Phage For This Age

Viral parasites of bacteria and other prokaryotic organisms, phage in general are extremely diverse in structure and genetic organization. Phage must attach to their specific prokaryotic host and ensure that their genomes (single or double-stranded DNA or RNA) enter the host cell. Usually the result of this is viral hijacking of the host and its conversion into a phage factory, ultimately leading to host cell destruction (lysis) and liberation of a large number of new phage particles. In broad outline, or course, this is a universal propagation strategy used by viral parasites across all kingdoms of life. Sometimes phage can be carried by their hosts indefinitely by a process termed lysogeny, where the phage integrates into the host chromosome or is carried as an independent replicating genome (plasmid replicon). Study of these and other features of phage biology has yielded a wealth of productive information. Owing to the relative simplicity of phage genomes, it is in fact no exaggeration to state that the rise of molecular biology itself derives largely from phage studies.

Phage have been major contributors to numerous biotechnological advances, and providers of information of general molecular biological significance. These viruses of prokaryotes can also be used as specific information sources potentially leading to novel therapeutics. In the case of pathogenic bacteria, systematic study of their phage has been shown to be a useful approach towards identifying phage-expressed proteins or peptides directed against bacterial host cell components. Identification of both the phage products and their host targets allows screening systems to be established for finding low molecular weight chemical inhibitors of the host cell targets themselves. Owing to the specificity of phage interactions with bacterial hosts, drug-modified phage have also been considered as vehicles for drug delivery to desired target cells. Modified phage have also been tested as vectors for transfection of mammalian cells with protein or nucleic acid ‘payloads’.

The use of bacteriophage in the never-ending war against bacterial pathogens does not stop with phage protein or nucleic acid components. Perhaps inspired by the dictum, ‘the enemy of my enemy is my friend’, phage themselves have long been considered as therapies for a variety of bacterial diseases. The idea is simple enough: kill infecting bacteria with their own parasites. Since phage are specific to their bacterial hosts and cannot replicate in eukaryotes, safety also is not likely to be a problem. Yet the history of ‘phage therapy’ has seen conflicting claims of efficacy, although many successful treatments have been reported. In recent times there has been renewed interest in the potential of phage therapy, accompanied by the rise in antibiotic-resistant bacteria and a slow-down in the pipeline of new replacement drugs. The great diversity of phage has been suggested to be superior to the diversity of natural antibiotics themselves. The unique feature of phage used in this manner is that they are self-replicating, indeed the first therapeutic agents in this category. Their capacity for self-replication in turn is directly linked with the population of their target hosts, which gives the pharmacokinetics of phage therapy some unusual aspects. It has been suggested that inadequate understanding of these novel pharmacokinetics is a major source of the mixed results previously seen with trials of phage therapy, and due attention to this detail may well shift phage therapy into a mainstream alternative to antibiotics. This is especially likely in cases of bacterial infections which are resistant to the current antibiotic armamentarium. Apart from direct therapeutic applications, phage have also been considered for environmental use in the reduction of bacterial loads in human-derived wastewater.

Another interesting feature of the exploitation of phage as whole viruses is that they are not a one-molecule ‘magic bullet’, and are not categorizable as ‘molecular solutions’ in a the sense of involving a single molecular entity. (Certainly a phage could not be deemed a ‘chrestomolecule’, although its subunit molecules could if they were of value in isolation). Phage often use elegant genetic systems for their regulation, and also show highly efficient and compact biological organization. (For example, the phage ΦX174 provided the first natural demonstration of overlapping genes). But despite such layers of underlying organizational complexity, phage have simplicity at the level of the types of their molecular constituents, all consisting of their informational nucleic acids (genomes) protected by a protein coat (capsid), combined with an entry system into their specific hosts. It then follows that the use of phage (whether natural or engineered) as therapeutic agents is an early example of exploitation of a (replicating) supramolecular system.

A huge abundance of as-yet uncharacterized phage are still to be found in the natural environment, and if past experience is anything to go by, such phage will provide rich fields of novel biodiversity. In an extension of the earlier overview (in the previous post) of the bounty  of marine bioproducts, marine bacteriophages have been cited as the largest reserve of untapped genomic information. In support of this contention is the observation that such phage have a very high proportion of unassignable open reading frames (predicted proteins which cannot be recognized as belonging to current known protein families). Current high-throughput sequencing technologies not only rapidly reveal new phage genes but allow high-altitude phage genomic comparisons, which are greatly facilitating studies of phage origins and evolutionary processes.

Other Viruses, or: What’s HIV Ever Done For Us?

Many viruses afflicting humans are infamous for the terrible toll they take on human lives, and for the resulting human suffering. Yet certain features of animal viruses can be harnessed towards desirable ends, especially their proficiency for targeting and invading specific host cells. As weapons against cancer, specific viruses have been modified and used to promote beneficial immune responses against cancers or infectious agents. Alphaviruses (a viral family which includes human encephalitis viruses) have been used as vectors to promote directed anti-tumor immune responses.  Alternatively, viruses can be designed to directly kill (lyse) tumor targets, with viral examples including herpesviruses and poxviruses. (Obviously, in all such cases, it is of great importance that the introduced genetic modifications inactivate the ability of the virus to damage normal cells).

Even the human immunodeficiency virus (HIV), the causative agent of AIDS, has had something to offer. As a member of the lentivirus family, HIV has the useful property of infecting non-dividing cells, which many other viruses cannot accomplish. Vectors have been derived from ‘gutted’ versions of the HIV genome which are unable to complete the usual replication cycle, but retain the ability to insert a copy of the viral RNA as a DNA sequence in the host genome. These features render them an attractive means for transferring desirable gene sequences to recipient cells of interest. For example, such lentiviral vectors have been used to transfer specific T cell receptors to T cells from human cancer patients. In such cases, the novel engineered T cell receptors are designed to recognize tumor-specific antigens, and allow the transduced T cells to then recognize and kill tumor targets. The lentiviral vector-mediated gene transfer process is also useful for gene therapy applications.

It is interesting that a pathogen as serious as HIV, the destroyer of tens of millions of human lives since its full-scale emergence in the early 1980s, can ‘give something back’ by way of contributing a useful system for human application. Not that this would compensate its victims, of course, but the very fact that anything positive can be gleaned from the HIV experience says something about the human ability to recognize natural opportunities when they arise. There are many other examples of ‘positives’ accruing from dangerous viral and bacterial agents of disease, but these have only been made possible through technology. In many forms, modern biotechnology provides both the means to perceive useful biological opportunities (whether from pathogens or otherwise), and also pathways towards their beneficial application.

Homage to Nature, and then Moving Beyond It

In the molecular domain, Nature’s bounty is translatable into human benefit at several levels, as we have seen. There is firstly of course the direct discovery and application of low molecular weight bioproducts (detailed in the previous post),  which has been done for thousands of years. The data provided by specific chrestobiomolecules can be used as a stepping-stone to even more effective agents, through successive rounds of human design intervention.

So, in the long run, the most important thing that large natural biomolecules and biosystems provide to us is information – information that acts as a spring-board towards the development of our own complex molecules and biosystems which are tailor-made towards specific applications. When this process is taken to its logical conclusion, we will reach a stage when we supersede the need to use such incredibly useful biological resources.

The use of higher-level systems in the future will thus inevitably become less a slavish derivation or copying of a natural precedent, and more and more an artificial improvement for specific design ends. This trend then increasingly overlaps with the field of synthetic biology, considered in several previous posts (30th  May, 7th June, 20th  June ).

Finally, a little comment on big molecules:

About natural products, one should recall:

They go far beyond molecules that are small

And think, if you please

That included in these

Are some considered the most useful of all.

References & Details

(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).

Many enzymes from a variety of biological sources are used in industrial processes…..’     See Kirk et al. 2002; Ahuja et al. 2004; Panke et al. 2004.

Certain bacteria thrive under amazing extremes of temperature, salinity, pH or pressure…..’     See Van den Burg 2003.

‘…..earth harbors an entire thermoresistant prokaryotic ecosystem…….’     See Gold 1992.

These [thermoresistant] organisms may be found in geothermal hot springs ….., at deep underground sites or at thermal vents in the deep oceans…..’     See Prieur et al. 1995; Deming & Baross 1993.

Thermophilic (heat-stable) enzymes in general have many applications….’     See Haki & Rakshit 2003.

‘….the polymerase chain reaction…’   For more details, see the accompanying ftp site for Searching for Molecular Solutions, for Chapter 4, Section No. 6 (‘PCR’).

At the other extreme from thermophilic organisms are psychrophiles……’ See Deming 2002;   ‘….and are also potential source of novel enzymes….’     See Cavicchioli et al. 2011.

‘….natural binding molecules produced ‘to order’ …….by diversification and selection mechanisms…..’    In brief, somatic diversification within immune cells of an vertebrate with an adaptive immune system results from combinatorial genomic rearrangements and somatic hypermutation, coupled with selection and amplification processes for cells uniquely expressing receptors recognizing a target antigen. This is also described (and referenced) in Chapter 3 of Searching for Molecular Solutions.

‘…..passive immunity has emerged as a therapeutic tool…..’     Among a vast number of examples can be found ‘antivenoms’ produced in horses or goats against various natural toxins from snakes, spiders, jellyfish, and other toxic organisms.

‘…..discovery of monoclonal antibodies…..’    See Köhler & Milstein 1975.

‘…….products based on antibodies have become a billion-dollar earner…..’    See Reichert et al. 2005.

‘…..antibodies can be raised such that they catalyze specific chemical reactions.‘    For some early references to this, See Pollack et al. 1986; Tramontano et al. 1986.

‘……Engineered T cell receptors in a number of forms have application….’   More details (and references) and provided in the Cited Notes for Searching for Molecular Solutions in the accompanying ftp site, for Chapter 7, Section 18, T Cell Receptor Technology and Applications.

‘….interferons, known initially for their antiviral properties, but also for their immunomodulatory activities….’    Modified isoforms of interferon-alpha have been widely used (in conjunction with antiviral drugs) for treatment of Hepatitis C (See Tsubota et al. 2011); interferon-beta has been used as a therapy for the autoimmune disease multiple sclerosis (See Rudick & Goelz 2011).

‘…..zinc-finger protein motif ….. ‘zinc finger nucleases’……’ For more recent information,    see Davis & Stokoe 2010; Carroll 2011.

‘…..development of protein frameworks……’    This is considered in Chapter 7 of Searching for Molecular Solutions.

‘……the rise of molecular biology itself derives largely from phage studies…..’   See Cairns, J., Stent, G. & Watson, J. Phage and the Origins of Molecular Biology. (Cold Spring Harbor Laboratory Press, 40th Anniversary Edition, 2007).

‘…….screening systems to be established for finding low molecular weight chemical inhibitors of the host cell targets themselves…..’    See Liu et al. 2004.

‘….drug-modified phage have also been considered as vehicles for drug delivery….’     See Yacoby et al. 2007.

‘……phage have also been tested as vectors for transfection of mammalian cells…..’     See Dunn 1996; Clark & March 2006.

‘……the history of ‘phage therapy’ has seen conflicting claims of efficacy…..’     See Sulakvelidze 2005.

‘……The great diversity of phage has been suggested to be superior to the diversity of natural antibiotics themselves…..’     See Schnaitman 2002.

‘…..the pharmacokinetics of phage therapy [has] some unusual aspects…….’; ‘……inadequate understanding of these novel pharmacokinetics is a major source of the mixed results….’  See Payne & Jansen 2003; Levin & Bull 2004.

‘…….phage have also been considered for environmental use…….’     See Withey et al. 2005.

‘…….the phage ΦX174 provided the first natural demonstration of overlapping genes…..’     When genes ‘overlap’, portions of their coding sequence occupy the same DNA tract, but in different reading frames. For the ΦX174 precedent, see Barrell et al. 1976.

‘…….marine bacteriophages have been cited as the largest reserve of untapped genomic information…….’    See Paul & Sullivan 2005.

‘……Current high-throughput sequencing technologies ……allow high-altitude phage genomic comparisons…..’    See Brussow & Hendrix 2002; Hendrix 2003.

‘……Alphaviruses …… have been used as vectors to promote directed anti-tumor immune responses.…..’     See Avogadri et al. 2010;   ‘…..viruses can be designed to directly kill (lyse) tumor targets (including herpesviruses…..’   See Todo 2008;  ‘……and poxviruses…..’   See Kirn & Thorn 2009.

‘…..lentiviral vectors have been used to transfer specific T cell receptors….’   See June et al. 2009;   ‘……also useful for gene therapy applications….’    See Dropulic 2011.

‘…..many other examples of ‘positives’ accruing from dangerous viral and bacterial agents of disease…..’   This general issue was discussed in the Searching for Molecular Solutions associated ftp site, in the Cited Notes for Chapter 3, Section No. 4 (DNA-binding protein design).

Note: If problems are encountered in directly accessing the above ftp site, go here, and then click on molecular-solutions.zip (8.11 Mb).

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