1 Recombinant protein scaffolds for tissue engineering Jerome A. Werkmeister and John AM ...

43 Pages·2011·0.45 MB·English

Recombinant protein scaffolds for tissue engineering

Jerome A. Werkmeister and John A.M. Ramshaw

CSIRO Materials Science and Engineering
Bayview Avenue, Clayton 3169, Australia

E-mail: [email protected]

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Abstract:
New biological materials for tissue engineering are now being developed using common
genetic engineering capabilities to clone and express a variety of genetic elements that allow
cost effective purification and scaffold fabrication from these recombinant proteins, peptides
or from chimeric combinations of these. The field is limitless as long as the gene sequences
are known. The utility is dependent on the ease, product yield and adaptability of these
protein products to the biomedical field. The development of recombinant proteins as
scaffolds, while still an emerging technology with respect to commercial products, is
scientifically superior to current use of natural materials or synthetic polymer scaffolds, in
terms of designing specific structures with desired degrees of biological complexities and
motifs. In the field of tissue engineering, next generation scaffolds will be the key to
directing appropriate tissue regeneration. The initial period of biodegradable synthetic
scaffolds that provided shape and mechanical integrity, but no biological information, is
phasing out. The era of protein scaffolds offers distinct advantages, particularly with the
combination of powerful tools of molecular biology. These include, for example, the
production of human proteins of uniform quality that are free of infectious agents and the
ability to make suitable quantities of proteins that are found in low quantity or are hard to
isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins
like collagens, elastin, silks and combinations of these, offers the further advantages of
natural well-defined structural scaffolds as well as the endless possibilities of controlling
functionality by genetic manipulation.

Keywords: Tissue engineering, scaffolds, recombinant proteins, fibrous proteins, collagen,
silk, elastin

Running Title: Recombinant protein scaffolds

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1. Introduction

Tissue engineering and regenerative medicine continue to engage a plethora of multi-
disciplinary scientists as an advanced strategy to restore or regenerate damaged or diseased
tissue. In its most simplistic form it comprises culturing autologous or allogeneic cells,
including stem cells, with or without appropriate cues and signals, in a range of scaffolds to
form functional tissues for implantation. The scaffold plays a pivotal part providing a
suitable support for cell adhesion, proliferation and in some cases differentiation. In addition
scaffolds can be fabricated to provide the necessary biochemical and biomechanical cues that
are required to mimic the natural extracellular matrix of the human body. The hope of
regenerative medicine is to ultimately treat patients in the most natural way that does not
simply add structural elements, but clearly is able to restore or induce tissue regeneration thus
avoiding the need for organ or tissue replacement (Williams and Sebastine 2005). Initial
approaches, which are still favoured by some, used synthetic polymers such as poly-lactic
acid and poly-lactic/glycolic acid copolymers as scaffold materials. While such scaffolds can
define the size and shape that is required from a scaffold construct, they present little
information to the cells on the type and complexity of the extracellular matrix that is required.
While adding growth factors and physical stimuli (Engler et al 2006) can assist in defining
the tissue that is formed, they are probably still insufficient to fully define and produce a
reasonable match to the native tissue that is being replaced. For example, to what extent do
tissue engineered heart valves match the complexity of collagen distributions that are found
in this tissue (White et al 2010) and which could be associated with the long-term durability
and effective performance of an engineered device.
Fibrous proteins are gaining increased attention in tissue engineering and, in
particular, the versatility in structure and design is making recombinant forms of these
materials the preferred option in advanced materials. Fibrous proteins are among the oldest
medical materials. Keratin, probably as horse or other hair, may have been used as an early
material and could possibly be the material mentioned for performing surgical suturing in the
earliest known medical text, 5000 BP, the Edwin Smith Papyrus (Breasted 1930). Silk would
undoubtedly have been used as an early medical material; for example, as sericulture,
especially using Bombyx mori, dating back to about 2700 BC, with other silk cultivation
possibly even earlier (Barber 1992). B. mori is the most well known silkworm, domesticated
from B. mandarina and used for silk production for over 5000 years. Legend has it that the
Chinese Princess Xi Ling Shi was the “true” inventor of the benefits of silk – the story is that
a silkworm cocoon fell into her hot water being prepared for tea and the silk thread began to
unravel itself (Wurm 2003). The oldest known suture is in a mummy from 1100 BC, while
the first detailed description of a wound suture and the suture materials used in it is by the
Indian physician Sushruta, written in 500 BCE. Development of collagen-based gut sutures
has been attributed to Galen who was cited as using them for suturing wounded gladiators
around 100 CE (MacKenzie 1973). The dissolving nature of catgut sutures was observed in
the 10th-century by Andalusian surgeon al-Zahrawi, who reportedly discovered the
dissolving nature of catgut when his lute's strings were eaten by a monkey. Within the last
century, collagen in particular has been used successfully in a wide range of medical
applications (Ramshaw et al 2009), perhaps more so than any other proteinaceous material.
Proteins, and especially fibrous proteins, typically have „high‟ information content.
The presence of different levels of information content in different cases of molecules has
long been recognised, especially by those working in the field of molecular evolution
(Zuckerkandl and Pauling 1965). The same concept is also true for molecules used as tissue
engineering scaffolds. While synthetic, repetitive polymers have little information content,
proteins with their structural complexity provide a wealth of potential information that cells
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can recognise and respond to. For example, type I collagen alone has more than 50
documented molecular interactions (Sweeney et al 2008), and the other collagen types, now
at least 28, contain even further content. Type IV collagen is a major component of basement
membranes and so has a different set of interactions than the interstitial collagens, including
interactions to different stem cell types. Although presently less numerous and less well
defined, elastin also has specific interactions, including the role of the C-terminal in cell
interactions (Kinikoglu et al 2011).
The availability of recombinant fibrous proteins goes back a long way (Werkmeister
et al 2003). For example, recombinant collagen production with hydroxylation of proline by
introduced prolyl-4-hydroxylase was initially described in 1993 (Prockop et al 1993),
although non-hydroxylated variants, including hybrid forms with other proteins were
described in the late 1980‟s (Williams et al 1988). Even then, obtaining hydroxylated
recombinant collagen in a commercial yield took around a further 10 years to achieve
(Neubauer et al 2007). Similarly, recombinant elastin was available in very low yield in
initial studies (Indik et al 1990), but it was only in 1995 that a system that could provide
commercial yields was devised (Martin et al 1995). This involved constructing a fully
synthetic gene of 2210 base pairs, in which the codons were optimised for expression in E.
coli. Equally, recombinant silk-like proteins were available a decade ago, but only as partial
constructs (Lazaris et al 2002), as full length constructs were too large and highly repetitive.
The recent advances in all these proteins, therefore, have occurred in refinement of
the various basic systems. Broadly, these include alternative production systems, designs
based on key structural elements, development of new sources, production of hybrid
structures with other proteins and composite materials, frequently with other fibrous proteins.
It is these refinements that are discussed in the following review.

2. Collagens

This section examines the production, efficiency and applications of recombinant collagens in
the biomedical industry. The precedent for use of recombinant proteins already exists in the
biopharmaceutical arena. The first recombinant protein biopharmaceutical, insulin, was
approved nearly three decades ago, and this had led to an exponential surge in the so-called
biotechnology-derived pharmaceutical products, largely of growth factors and humanised
monoclonal antibodies. It is expected that the same tools can be used to engage in the
biomedical and tissue engineering fields to open new pathways and systems to treat damaged
or diseased tissues.

2.1 Background

Collagens are the major protein of all connective tissues comprising a family of 28
genetically distinct types (Ramshaw et al 2009), each characterised by a common triple
helical structure that forms the building block of tissue architecture. Collagen type I is the
predominant interstitial fibril-forming collagen and for this reason has been the easiest and
most abundant source for use as biomaterial and medical devices (Ramshaw et al 2009). All
collagens are defined by 3 helical chains, each in a left handed polyproline II-like helix, that
wind together to form a right handed super-coiled triple helix. At the molecular level it is
defined by a unique three amino acid repeat, (GXY), where glycine is always found in every
third position, and the Xaa and Yaa positions are often occupied by the imino acid proline. In
the Yaa position the proline is modified post-translationally to 4-hydroxyproline (Hyp),
which confers stability to the triple helix, and enables collagens, at least mammalian
collagens, to be thermally stable at human body temperature.
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The biosynthetic collagen pathway is quite complex and requires a number of
secondary essential modification steps including some that are important for crosslinking and
helical stability. For recombinant expression of collagen type I or any collagen, it is essential
to mimic the part of the natural biosynthetic pathway that confers stability. In most
expression systems this requires the co-expression of prolyl-4-hydroxylase (P4H; E.C.
1.14.11.2) which is translated from two genes that produce an active tetramer α β of P4H
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that is essential for hydroxylation of proline in the Y position leading to helical stability.

2.2 Expression of mammalian collagen

Almost every possible type of expression system has been used for recombinant expression
of collagen, collagen-like proteins and selected domains. Largely, the wide array of
expression systems has been used to further enhance the understanding of collagen structure-
function in normal and pathological conditions. But the availability of gene sequences from
all collagen types adds certain value to the future of using some of these as added value
products for specific applications. For instance, collagen type XXVIII, a novel von
Willebrand factor A domain-containing protein, is highly localised in the sciatic nerve at the
basement membrane of certain Schwann cells surrounding nerve fibres (Veit et al 2006);
collagen type XXII exhibits a strikingly restricted localization at tissue junctions, particularly
the myotendinous junction in skeletal and heart muscle (Koch et al 2004); collagen type XVII
is a transmembrane constituent of the epithelial anchoring complex involved in maintenance
of cell adhesion at this interface (Van den Bergh et al 2006); collagen type XV, structurally
homologous to collagen type XVII abundant in liver, is highly expressed in the skeletal
muscle and regulates cell adhesion and migration (Hurskainen et al 2010).
For commercial applications, as medical grade products for biomaterials or scaffolds
in regenerative medicine, the recombinant approach offers distinct advantages including:
availability and uniformity of product quality; ease of isolation and purification in certain
systems; freedom from infectious agents associated with current animal-derived products;
potential to produce any of the rare collagen types, not just the routine collagen type I;
potential scope to modify and improve on natural protein structures, including selection of
specific domains, engineering products with multiple advantageous repetitive motifs or
chimeric molecules.
In addition, cost-effectiveness is the key factor that will decide the future of these
proteins. Recombinant collagens are now available commercially largely for in vitro
applications using a very precise expression system aimed at low cost with reasonable yield
(Baez et al 2005).

2.2.1 Prokaryotic expression
E. coli is the most common bacteria used for expression of recombinant proteins including
some extracellular matrix proteins. It is possible to use bacterial expression systems for small
motifs, particularly those with inherent capacity to re-fold. There are multiple issues with
this approach in general, at least for mammalian collagen proteins. Firstly it requires co-
expression of the P4H enzyme which has been very problematic in bacteria; active P4H
tetramer has been occasionally expressed in E. coli using a special strain of bacteria with a
high oxidising cytosol (Neubauer et al 2005). In addition, the nature of these proteins means
that most will end up as insoluble denatured proteins (inclusion bodies) within the bacteria.
Production of full length mammalian collagen is highly unlikely in this system and most
products reflect the denatured form of this protein, gelatine (Ferrari et al 1993, Cappello
1998). Smaller mammalian collagen fragments have been produced with limited success; for
example, part of the collagen type V α1 chain comprising the heparin binding site (Ruggiero
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and Koch 2008) has been expressed as a soluble functional small motif and production yields
were increased using strain of bacteria that could be induced with salt rather than the standard
lac promoter inducer IPTG (Ricard-Blum et al 2006).
More recently, there have been a number of scattered reports on optimisation of E.
coli for enhanced biomass production of recombinant human-like collagens. These include
enhanced growth and expression by controlled CO pulsing (Xue et al 2009), increasing the
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fermentor pressure and elevating the oxygen transfer rate (Ma et al 2010), medium
optimisation of the carbon/nitrogen ratio (Guo et al 2010) and assimilation of acetic acid
production by glucose starvation (Xue et al 2010). In these limited studies from these
Chinese groups, the increase in biomass and product yield has been significant. With the
control of oxygen transfer methods, the cell density and volumetric product yield increased
by 2-fold to 77 gl-1 and 14 gl-1 respectively; with the medium optimisation methods, optimal
carbon/nitrogen molar ratios for batch (2.36:1) and feeding media (5.12:1) yielded similar
biomass of 67.2 gl-1 and collagen production of 10.8 gl-1. In all these studies it still remains
questionable if the product is indeed recombinant collagen or merely gelatin as no evidence is
given to the precise characterisation of the homotrimeric collagen type II that was produced.

2.2.2 Eukaryotic expression
The lack of effective post-translational processing of collagens in bacteria has favoured a
number of eukaryotic systems for collagen production. The choice here is large and there are
certain advantages and disadvantages for each system. Certainly the simplest approach is to
use a system that does not involve the co-expression of the essential P4H enzyme.
Mammalian cell lines, including CHO, COS, HEK 293and HT1080, offer this distinct
advantage – they all have the complete post translational machinery for correct folding and
stability of collagen. Indeed given the complex biosynthetic pathway for collagen synthesis,
it is not surprising that mammalian expression systems have always been the first choice for
collagen production for purely scientific discovery and understanding collagen behaviour.
The downside, however, is the very poor yield, generally less than 0.1-0.5 mgl-1 (Ruggiero
and Koch 2008). The HEK 293-EBNA cell line was developed as an optimised mammalian
expression system with very low background matrix production. This cell line has been used
to express larger amounts of a number of matrix proteins including collagen types V, VII,
VIII, X, XVI. Production yields have increased up to 80 mgl-1. Large scale production of
another matrix protein, the globular LG4/5 domains of laminin 5, has been reported to be
much higher, around 3 gl-1, using this system (Belin and Rousselle 2006). While offering a
simple and easy approach to producing stable collagen, this methodology is not suitable for
commercial production due to low yields.
The most common expression system for large scale mammalian collagen production
is yeast. While mammalian cells have P4H activity and insect cells have poor intrinsic P4H
capacity, yeast cells do not possess this enzyme and so expression systems have been
optimised to integrate and co-express at least 3 concurrent genes (at least 1 collagen gene for
homotrimeric collagens and 2 genes for the P4H). Saccharomyces systems can be used and
various designs have been adopted to integrate the 3 genes necessary for stable collagen
production (Vaughan et al 1998, Toman et al 2000). Most of these studies, as well as those
described below in Pichia systems, have been focused on large scale production of
unmodified recombinant collagens. From a tissue engineering aspect, it is important to use
these systems to optimise the genetic design to enhance performance. Fertala and colleagues
have engineered a gene product comprising multiple repeats of a biological active complete
D4 period (Majsterek et al 2003). We have recently shown it is possible to design and
incorporate much smaller specific biological motifs to direct cellular function, and cell
function can be enhanced by constructing genes with 2 or more repeating motifs encoding
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these biological entities (Peng et al 2009). While product yields are still low in this system,
these and other expressed proteins can be used to coat or add value to other types of
scaffolds.
P. pastoris is the most well developed yeast strain currently developed for effective
commercial collagen production. It has been successfully engineered to co-express the P4H
with the appropriate mammalian collagen gene. Fermentation systems have seen
improvements of protein yield from 0.015 gl-1 to 1.5 gl-1 or greater for full length collagen,
and up to 14 gl-1 for smaller defined collagen fragments (Baez et al 2005). There have been
various approaches to improve biomass and product yields and it is the combination of all
these, rather than one single improvement that has driven commercial viability. These
include codon optimisation for P. pastoris; deletion of the N-propeptide which shortened the
gene length and which was found to be unnecessary for correct folding; replacement of the C-
propeptide with a shorter trimerizing foldon peptide from the phage T4 fibritin; optimisation
of integration and copy number for P4H; fermentation at a reduced temperature of 32° C
which increased likelihood of proline hydroxylation in the Y position; oxygen enrichment to
increase the P4H activity that requires high concentrations of molecular oxygen; high
biomass and subsequent product accumulation by designing an animal free fermentation
broth with an initial glycerol feed containing hexametaphosphate and ammonium sulphate.
Of all available yeast strains, P. pastoris has the potential for the highest expression levels
with appropriate post-translational modifications. The system uses a strong promoter of
alcohol oxidase (AOX1) which, after the initial glycerol feed and biomass optimisation, is
induced by methanol which also acts as the sole carbon energy source. The combination of
all these factors have led to high end point product yields (Baez et al 2005). This has
involved patient and precise improvements along the way, but also initial upfront engineering
costs to safely design large scale flame-proof fermentation facilities for production using high
volumes of oxygen and methanol. Generally, optimisation of bioreactors uses simple fed-
batch fermentation with controlled continuous addition of methanol. Improved production of
human type II procollagen has recently been reported in P. pastoris in simple shake flasks
using wireless-controlled fed batch (Ruottinen et al 2008), resulting in a 10-fold higher
amount of mRNA and protein synthesis. Hansenula polymorpha, now re-classified to the
Pichia genus, offers an alternate yeast strain that has been reported to have endogenous P4H
activity and could possibly be used as a simpler expression system (de Bruin et al 2002). In
this initial report only very small collagen fragments were expressed and some of the proteins
may have been complicated by an endogenous collagen in the organism itself. A more recent
study was unable to generate hydroxylated collagen and in addition failed to identify a gene
encoding the P4H in the genome (Geerlings et al 2007).
Downstream processing of fermented organisms need to be considered – generally
these have to be simple, compliant with regulation and cost effective. For Pichia systems, the
process is exactly that – the purification system does not involve laboratory chromatographic
methods. Cross flow filtration, enzyme digestion and differential salt precipitation at defined
pH is usually the method of choice.
The issue for production of heterotrimeric collagen types is still challenging.
Collagen type II and III are homotrimers and there have been no issues with production of
thermostable products. But for collagen type I there is a preference to assemble the non-
natural [α1(I)] homotrimer and not the natural heterotrimeric [α1(I)] α2(I) in the Pichia
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system (Baez et al 2005). This, however, may not be unique to yeast systems as it appears to
be a general phenomenon associated with heterologous expression systems. In a recent
report, Roulet and colleagues used collagen type V as a model collagen product (Roulet et al
2010). Collagen type V, a fibrillar collagen like type I, II and III collagen, is naturally found
as an abundant ubiquitous heterotrimeric collagen, [α1(V)] α2(V) and a less common
2
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homotrimer form, [α1(V)] Surprisingly, using yeast, insect and even mammalian HEK-293
3.
cells, the formation of the homotrimer [α1(V)] was considerably favoured over the
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heterotrimer. There is only one company, Fibrogen (www.fibrogen.com/collagen) that
produces highly purified fully characterised recombinant collagens for laboratory use and
intended for medical applications.

2.2.3 Use of transgenic plants and animals
A transgenic animal or plant is one that contains a deliberate modification to the genome such
that the foreign introduced DNA is transmitted through the germ line to every cell. The
simplest way of doing this is by using conventional recombinant DNA tools to microinject
the foreign DNA directly into an ovum.
Several transgenic animal species are capable of producing recombinant proteins from
milk, blood, egg white, semen, urine and silk glands (Houdebine 2009). The mammary gland
is the most explored system to express many recombinant proteins in transgenic animal
bioreactors, followed by chicken egg white. Quite a number of mammalian species can be
used including pigs, sheep, goats, cows, rabbits and mice. Each will have its own advantages
and disadvantages. From a cost effective exercise, pigs or rabbits are seen to be the choice
due to high levels of milk, high fertility and disease-resistance, although other options like the
dwarf Nigerian goat have also been attempted for production of silks.
There has been very little work on use of animal transgenics for mammalian collagen
production. Expression of foreign genomic human α1(I) in mouse milk resulted only in
severely under-hydroxylated procollagen that was thermally unstable suggesting inadequate
endogenous P4H (John et al 1999). With the introduction of foreign α and β genes of P4H
into the genome, reasonable levels of stable collagen, upto 0.2 gl-1 were produced (John et al
1999). A comparable report (Toman et al 1999, Berg 2000) produced much higher
production yields of stable collagen around 8 gl-1 but these were truncated 37Kb molecular
weight proteins and again all were homotrimeric in nature.
While the use of animal transgenics has not been followed up from these early
reports, the use of transgenic plants does offer some cost effective and commercial outcomes
(Dyck et al 2003). Transgenic plants have a track record as bioreactors for pharmaceuticals
(eg insulin, lactoferrin, and antibodies), vaccines, industrial processing enzymes (trypsin)
largely due to the belief that these systems offer practical and cost effective solutions to
recombinantly-expressed proteins.
The first reports on transgenic expression of recombinant collagen-related proteins in
plants used a model system of tobacco (Ruggiero et al 2000). These tobacco plants were able
to produce fully processed triple helical human collagen type III (Olsen et al 2003) and
collagen type I homotrimer (Ruggiero et al 2000). Of particular significance in these studies,
was that while tobacco plants did provide a useful expression system for large scale
production of recombinant collagen, hydroxylation of proline in the Y position did not occur
effectively. So while there was some degree of triple helical folding occurring that allowed
clues on the role of hydroxyproline in collagen folding and fibrillogenesis (Perret et al 2001),
collagen expression in transgenic plants does require the additional genomic insertion of the
genes for the P4H. Barley has also been used to produce full length collagen type I α chain
by targeting to the endoplasmic reticulum using 3 different promoters (maize ubiquitin,
endosperm-specific glutelin or a germination-specific α-amylase) (Eskelin et al 2009). In this
study and others using Wave bioreactors to culture transgenic barley cells (Ritala et al 2008),
the highest yields were obtained with the glutelin promoter (140 mg/kg seed) which is
relatively still very low. More recently tobacco plants have again been tried as transgene for
production of heterotrimer collagen type I (Stein et al 2009). In this study both collagen α
chains were co-expressed with both genes encoding the P4H enzyme as well as the gene
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encoding lysyl hydroxylase. Plants co-expressing all 5 vacuole-targeted proteins resulted in
generation of full length collagen with extremely high yields around 2% (20 gl-1) of the
extracted total soluble protein. This approach using tobacco for large scale collagen
production has now been recently commercialised by CollPlant Ltd (www.meytavti.co.il).
Transgenic corn has been extensively studied for the large scale production of
recombinant industrial and medical pharmaceuticals. Corn, like other plant crops, offers
advantages of large volumes (1,000‟s tons per year) and low cost around US $50/kg of grain
(Zhang et al 2009a). Corn-grain production of a small human α1 collagen fragment (44 kDa)
showed around 18% hydroxylation (2% hydroxyproline compared to maximum 11%
expected in animal collagen type I) suggesting some endogenous P4H activity but the yields
were only around 3 mg/kg (Zhang et al 2009a). Comparable low yields and low level
hydroxylation also occurred with full length recombinant collagen type I using embryo
specific promoters and a foldon to enhance trimerization (Zhang et al 2009b). The issue of
improving product yields is currently under investigation and largely reflects methods of
optimising protein extraction in a simple and cost effective approach without the cumbersome
traditional analytical methods of purification (Zhang et al 2009b, Paraman et al 2010). Both
dry and wet milling approaches are being optimised – both seem to be compatible with large
scale up and purification. Dry milling is simple and can be performed on the farm
minimising cost and potential inadvertent release; wet milling is largely more effective in
yields and purity
Transgenic silkworms have also been investigated as a convenient bioreactor system
for production of recombinant collagen and chimeric products. Many recombinant proteins
have been produced, particularly over the last decade, in silkworm larvae or pupae (Kato et al
2010). The benefits of transgenic silkworms is the convenience and cost effectiveness with
increase product yields in most cases ranging from 20 to 10,000 fold compared with
laboratory methods. Using a piggyBack transposon-derived vector system, recombinant
collagen type III has been successfully produced in a stable form in the silk glands of B.mori
larvae (Tomita et al 2003). The global annual production of silk is around 60,000 tons so the
mathematics and logistics of this approach for foreign protein production is positive. As an
additive to the natural silk product to improve cell function, the fibroin light chain gene from
B. mori has been modified with a collagen insert - an 8 repeat of a 10 triplet GXY sequence
with a C-C at the C-terminal end for stability (Yanagisawa et al 2007) which enhanced cell
adhesion. More recently, full length collagen type I α chain has been produced in the middle
silk glands and secreted into the cocoons of transgenic silkworms (Adachi et al 2010). The
initial yields were poor (0.8%), but was improved significantly to around 8% (4.24 mg per
cocoon) by cross breeding with moths carrying a gene of the transactivator IE1 from
baculovirus. CD spectra and thermal transition curves show a lack of native triple helical
structure, but the product was still capable of supporting cell growth; in particular the
recombinant substrate was capable of supporting Cynomologous monkey embryonic stem
cells in their undifferentiated state for a remarkable 30 passages.

2.3 Bacterial collagens

Proteins comprising the repeating triplet of GXY with high proline content have the potential
to form a left handed polyproline II and, with 3 chains, can then form a typical right-handed
triple helical structure (Ramachandran 1988). Collagens are already known to be present in
non-eukaryotes like sponges, mussels and worms. To some extent then, there was no great
surprise that DNA encoding these collagen-like sequences was also found in the genome of
bacteria (Ferretti et al 2001) and bacteriophages (Smith et al 1998). What was a surprise is
that these genes can produce thermally stable collagen proteins even though these organisms
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are unable to synthesize hydroxyproline (Figure 1). A more thorough bioinformatics BLAST
search of available bacterial genome databases (137 eubacterial genomes; 15 archaebacterial
genomes; 30 genomes of lower eukarya and against viral genomes) identified 103 collagen-
related structural motifs (CSM) containing GXY repeats (Rasmussen et al 2003). The length
of these CSMs varied from 7 to 745 continuous triplet repeats with a mean length of 76 GXY
repeats. In particular, 53 CSMs were found from the 137 eubacterial genomes examined, and
these contained a significantly higher proportion of threonine and glutamine in the Y position
with a propensity for the X position to be proline, alanine or serine. The other important
trend was the uneven distribution of charged residues; most frequently the X positions having
a negative charge, the Y positions a positive charge. Molecular modelling suggested that the
threonine in the Y position is able to form intermolecular hydrogen bonds to near-by carbonyl
groups in the helix backbone, and the concept of charge, ionic pairing and sequence stability
has been validated theoretically and experimentally (Persikov et al 2005).
There is a large group of pathogenic bacteria that express these collagen-like
sequences that are largely associated with host colonization and virulence in humans,
including Group A Streptococcus (Lukomski et al 2000), Bacillus anthracis exosporium
(Sylvestre et al 2002) and in insects, for example, the fungal pathogen Metarhizium
anisopliae (Wang and St Leger 2006). Depending on the bacteria strain, the Group A
Streptococcus presents multiple proteins including the streptococcal collagen-like proteins
Scl 1(Lukomski et al 2000) and Scl 2 (Lukomski et al 2001), the former comprising the
GLPGER sequence which can interact with the α β and α β integrins and induce
2 1 11 1
intracellular signalling (Humstoe et al 2005) or can interact with other extracellular matrix
proteins including fibronectin and laminin (Caswell et al 2010). In a more in depth study,
Caswell and colleagues used the “blank slate” from a non-cell binding integrin-lacking CSM
sequence from Scl2.28 to introduce various G-Xaa-Yaa sequences to confirm the identity of
the prokaryotic sequence for the human collagen receptors and the higher affinity for the
α β integrin (Caswell et al 2008). There may be some immunological concerns with these
11 1
bacterial collagen-like proteins. By the nature of their natural function, these proteins could
be thought to immunodominant proteins in their natural form as bacterial antigens. The
collagen-like protein from B. anthracis exosporium can induce antibodies in mice with
adjuvant and can further act as a booster to help protect mice against suboptimal amounts of
infecting bacteria (Brahmbhatt et al 2007); these antibodies, however, did not cross react
against human collagen types I, III or V. In a more recent study we have shown that the
recombinant Scl2 protein is not immunogenic in mice either with or without the presence of
adjuvant (Peng et al 2010).
The presence of GXY repeats is also predicted in non-pathogenic bacteria
(Rasmussen et al 2003) and recombinant proteins from a mixture of Gram-positive and
Gram-negative bacteria (for example Methylobacterium sp 4-46, Rhodopseudomonas
palustris and Solibacter usitatus) have been expressed in E .coli and characterized (Xu et al
2010). All of the expressed products were soluble and maintained similar triple helical
thermal stability irrespective of the diverse variation in amino acid sequences. In the same
study, the CSM from Clostridium perfringens was insoluble but could be coaxed into
secreting soluble thermally stable products by flanking the CSM with heterologous folding
trimerization domains at either the N or C-terminus. Assembly of stable human type I and III
collagen molecules in P. pastoris has been shown to be dependent on the efficiency of
trimerization; the substitution of the simple foldon sequence, derived from the native T4
phage fibritin, was able to generate 2.5 to 3-fold increase levels of collagen expression
compared with the natural C-propeptides (Pakkanen et al 2003). The foldon sequence has
also been used in E. coli expression systems to augment triple helical stability of collagens
(Du et al 2008); in this instance the authors claim to express stable human collagen products
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