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Bioengineered collagens a
a
John AM Ramshaw , Jerome A Werkmeister & Geoff J Dumsday
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CSIRO Materials Science and Engineering; Clayton, Australia Published online: 09 Apr 2014.
Click for updates To cite this article: John AM Ramshaw, Jerome A Werkmeister & Geoff J Dumsday (2014) Bioengineered collagens, Bioengineered, 5:4, 227-233, DOI: 10.4161/bioe.28791 To link to this article: http://dx.doi.org/10.4161/bioe.28791
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
REVIEW
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Bioengineered 5:4, 227–233; July/August 2014; © 2014 Landes Bioscience
Bioengineered collagens
Emerging directions for biomedical materials John AM Ramshaw*, Jerome A Werkmeister, and Geoff J Dumsday CSIRO Materials Science and Engineering; Clayton, Australia
Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications.
Recombinant Collagen: Establishing Commercial Availability Collagen is the most abundant protein in mammals, where it plays critical roles in the structure and in molecular and cellular interactions in the extracellular matrix. For example, collagen is the major protein of many tissues, including skin, bone, ligament, tendon, and cartilage. Collagens are characterized by a unique molecular structure, the collagen triple-helix. This structure consists of a supercoiled triple-helix, which is made from three left-handed polyproline-like chains twisted together into a righthanded triple-helix.1 A further, interesting feature of this structure is that the tight packing of the triple helix requires that every third residue in the primary sequence be Gly, because there is no space for any larger amino acid in the interior axis of the triple-helix. As a consequence, a second characteristic feature of collagens is the repetitive amino acid sequence pattern (Gly-Xaa-Yaa) n.1 A further feature, is that the non-Gly positions are often occupied by the imino acid proline. For animal collagens, Pro residues in the Yaa position, about 10% of all residues, are normally posttranslationally modified by the enzyme prolyl-4-hydroxylase *Correspondence to: John AM Ramshaw; Email: [email protected] Submitted: 02/19/2014; Revised: 04/03/2014; Accepted: 04/04/2014; Published Online: 04/09/2014 http://dx.doi.org/10.4161/bioe.28791
(P4H), to give 4-hydroxyproline (Hyp). This modification is very important for several reasons, particularly as an absolute requirement for good thermal stability of the triple-helix, but also to assist in self association, especially in the fibrillar collagens, and participation in certain receptor interactions.1,2 Although collagen is used as a single all-embracing term, in humans and other mammals, there are at least 28 genetically distinct types,2 all of which exemplify these various structural characteristics. The most abundant collagens are the interstitial, fibril forming collagens, particularly type I collagen, which are present in all the major connective tissues (Fig. 1). Type I collagen, which can be readily obtained and purified, has become the major collagen used in biomedical and tissue engineering applications. Although there are some early examples, it is only in the past 100 years that a range of collagen-based biomedical materials have emerged, starting from the use of human amnion in 1912, followed by collagen based dressings in the 1940s,3 with many subsequent products.4 Most recently, there has been a growing interest in using natural extracellular matrix-based, particularly collagen, supporting structures (scaffolds) in tissue engineering developments.5 In all these cases of medical applications, there are a number of concerns that have been expressed. These include, for example, the natural variability in preparations from animal tissues, where purity and predictability of performance are issues. Of more concern has been the possibility of transmission of animal based diseases, especially bovine spongiform encephalopathy (“madcow disease”). There have been two suggested approaches to this disease issue. One approach has been the examination of non-mammalian species, including jellyfish, fish, and chicken as material sources,6 while the other approach has been the production of collagen by recombinant technology.7 Recombinant approaches enable production of a disease-free product that is of uniform product quality. The approach also could allow for the production of less common collagen types, where extraction from tissue is not practical. There is also the opportunity to modify and improve on the natural protein structures by introduction of mutated residues, selection of specific domains, engineering products with multiple repetitive motifs, and development of chimeric structures.7 The potential disadvantage of recombinant technology, however, is that recombinant expression of type I collagen, or any mammalian collagen, will almost certainly also need the co- expression of functional P4H to enhance the triple-helical stability. The P4H enzyme consists of two different chain types,
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Keywords: collagen, recombinant expression, triple helix, thermal stability, prokaryote, biomedical material, tissue engineering
each with a molecular mass of around 65 kDa. If the stability of the collagen triple-helix is low, then the product is the denatured form of collagen, gelatin. Gelatin consists of only single chains that are not folded into in the native triple-helical structure. Also, while recombinant technology can be used easily for small scale production of laboratory samples, for commercial scale production there will be manufacturing issues that will need to be solved and process economics evaluated.8 Initial biotechnology developments focused on the production of full-length human collagens, including the heterotrimeric type I collagen and the homotrimeric type III collagen.7 Mammalian cell systems, such as HT1080 and HEK 293-EBNA cells, have been used, but yields are very low. The same is also true for production in an insect cell system, Baculovirus. Both systems, however, do have endogenous P4H activity (Table 1). Microorganisms provide alternative expression systems and are a natural choice because of their familiarity in the biotechnology industry.9 Unfortunately, the use of E. coli expression was initially not seen as practical, as there seemed to be difficulties in co-expressing all the necessary genes.7 On the other hand, yeast systems were considerably more promising, and several have been used successfully for the expression of full-length hydroxylated human collagens (Table 1).10-12 The advantage of the yeasts is they can be optimized to integrate and co-express 3 or 4 genes concurrently. The most successful application, which has provided commercial opportunities, has emerged from expression in P. pastoris (Table 1).11 Yeasts, as with other microorganisms typically require different codon preferences to those in mammalian systems for optimal expression. The development of rapid gene synthesis enables effective codon optimized sequences as well as specific variants of these sequences to be readily produced resulting in giving good yield.
228
Recombinant Bacterial Collagen: An Emerging System Collagens were seen historically as being associated with multicellular animal tissues. In more recent times the suggestion of collagen-like molecules in other species has emerged, with (Gly-Xaa-Yaa)n repeating sequences present in a few fungi,20 in viruses,21 and in phage.22 Most interesting, however, are the numerous collagen-like structures that are being identified in bacteria and in the emerging, large numbers of bacterial genomes. Thus, a gene with a collagen-like repeat, that is a virulence factor, was identified and characterized in the bacterium Streptococcus pyogenes.23 Subsequently, a second, structurally distinct, collagenlike gene was identified from the same species.24 Both were shown by biochemical and biophysical studies to have the characteristic collagen triple-helical structure.25 The surprise that emerged was that these collagens were both stable around mammalian body temperature, with melting temperatures of 36.4 °C and 37.6 °C, without the presence of any hydroxyproline.25 A decade ago, Rasmussen and colleagues reported an analysis of the available bacterial genome databases, searching for collagen-related structural motifs (CSM).26 The study included 137 eubacterial genomes and 15 archaebacterial genomes and looked for sequences with homology to (Gly-Pro-Pro) n where n was 7 or more. This identified 53 proteins in 25 bacterial
Bioengineered
Volume 5 Issue 4
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UNAM Ciudad Universitaria] at 03:01 05 January 2015
Figure 1. A transmission electron micrograph of the collagen fibrils in a sheep anterior cruciate ligament, showing numerous collagen fibrils, all of similar diameter. Bacterial collagens do not form such fibrillar structures. Bar = 0.25 µm.
Other approaches to improve commercial yields are also possible and include modification of the propeptide domains, enhanced integration, increased copy number of introduced genes, and reduced production temperature and oxygen enrichment to increase the P4H activity. Other systems have also emerged, based on transgenic organisms. For collagens, the mammary gland has been used for collagen production in mouse milk (Table 1).13 Another, more unusual system that is suited for fibrous proteins has been production of recombinant human type III procollagen in cocoons of transgenic Bombyx mori silkworms.14 Collagen production in transgenic plants has also been developed and seems to provide cost-effective, commercial outcomes. Initial reports produced unhydroxylated or poorly hydroxylated collagen from leaves15 and from seeds.16 Additional introduction of the mammalian P4H is necessary for a quality collagen product, even though plants contain a form of P4H. Most recently, heterotrimeric collagen has been produced with P4H also present, giving excellent protein yields, which can be commercialized.17 There may be further advantages of recombinant systems that possibly work only at a laboratory level. For example a range of constructs have been made for type II collagen, where specific “D-period” repeats, about 234 amino acids each, have been removed or substituted into the structure, allowing a better understanding of collagen structure and function.18 Another variation to this approach has been the design and production of small collagen fragments with embedded multiple repetitive domains (integrin) within the collagen chain.19
Table 1. Examples of recombinant systems for human collagen production * Production systems
Yield
Comments
HT1080
≤1 mg/L
Mainly type II collagen and variants
HEK293
≤80 mg/L
Used to produce collagen types V, VII, VIII, X, XVI
CHO
Bioengineered Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kbie20
Bioengineered collagens a
a
John AM Ramshaw , Jerome A Werkmeister & Geoff J Dumsday
a
a
CSIRO Materials Science and Engineering; Clayton, Australia Published online: 09 Apr 2014.
Click for updates To cite this article: John AM Ramshaw, Jerome A Werkmeister & Geoff J Dumsday (2014) Bioengineered collagens, Bioengineered, 5:4, 227-233, DOI: 10.4161/bioe.28791 To link to this article: http://dx.doi.org/10.4161/bioe.28791
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
REVIEW
Paper Type
Bioengineered 5:4, 227–233; July/August 2014; © 2014 Landes Bioscience
Bioengineered collagens
Emerging directions for biomedical materials John AM Ramshaw*, Jerome A Werkmeister, and Geoff J Dumsday CSIRO Materials Science and Engineering; Clayton, Australia
Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications.
Recombinant Collagen: Establishing Commercial Availability Collagen is the most abundant protein in mammals, where it plays critical roles in the structure and in molecular and cellular interactions in the extracellular matrix. For example, collagen is the major protein of many tissues, including skin, bone, ligament, tendon, and cartilage. Collagens are characterized by a unique molecular structure, the collagen triple-helix. This structure consists of a supercoiled triple-helix, which is made from three left-handed polyproline-like chains twisted together into a righthanded triple-helix.1 A further, interesting feature of this structure is that the tight packing of the triple helix requires that every third residue in the primary sequence be Gly, because there is no space for any larger amino acid in the interior axis of the triple-helix. As a consequence, a second characteristic feature of collagens is the repetitive amino acid sequence pattern (Gly-Xaa-Yaa) n.1 A further feature, is that the non-Gly positions are often occupied by the imino acid proline. For animal collagens, Pro residues in the Yaa position, about 10% of all residues, are normally posttranslationally modified by the enzyme prolyl-4-hydroxylase *Correspondence to: John AM Ramshaw; Email: [email protected] Submitted: 02/19/2014; Revised: 04/03/2014; Accepted: 04/04/2014; Published Online: 04/09/2014 http://dx.doi.org/10.4161/bioe.28791
(P4H), to give 4-hydroxyproline (Hyp). This modification is very important for several reasons, particularly as an absolute requirement for good thermal stability of the triple-helix, but also to assist in self association, especially in the fibrillar collagens, and participation in certain receptor interactions.1,2 Although collagen is used as a single all-embracing term, in humans and other mammals, there are at least 28 genetically distinct types,2 all of which exemplify these various structural characteristics. The most abundant collagens are the interstitial, fibril forming collagens, particularly type I collagen, which are present in all the major connective tissues (Fig. 1). Type I collagen, which can be readily obtained and purified, has become the major collagen used in biomedical and tissue engineering applications. Although there are some early examples, it is only in the past 100 years that a range of collagen-based biomedical materials have emerged, starting from the use of human amnion in 1912, followed by collagen based dressings in the 1940s,3 with many subsequent products.4 Most recently, there has been a growing interest in using natural extracellular matrix-based, particularly collagen, supporting structures (scaffolds) in tissue engineering developments.5 In all these cases of medical applications, there are a number of concerns that have been expressed. These include, for example, the natural variability in preparations from animal tissues, where purity and predictability of performance are issues. Of more concern has been the possibility of transmission of animal based diseases, especially bovine spongiform encephalopathy (“madcow disease”). There have been two suggested approaches to this disease issue. One approach has been the examination of non-mammalian species, including jellyfish, fish, and chicken as material sources,6 while the other approach has been the production of collagen by recombinant technology.7 Recombinant approaches enable production of a disease-free product that is of uniform product quality. The approach also could allow for the production of less common collagen types, where extraction from tissue is not practical. There is also the opportunity to modify and improve on the natural protein structures by introduction of mutated residues, selection of specific domains, engineering products with multiple repetitive motifs, and development of chimeric structures.7 The potential disadvantage of recombinant technology, however, is that recombinant expression of type I collagen, or any mammalian collagen, will almost certainly also need the co- expression of functional P4H to enhance the triple-helical stability. The P4H enzyme consists of two different chain types,
www.landesbioscience.com Bioengineered
227
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UNAM Ciudad Universitaria] at 03:01 05 January 2015
Keywords: collagen, recombinant expression, triple helix, thermal stability, prokaryote, biomedical material, tissue engineering
each with a molecular mass of around 65 kDa. If the stability of the collagen triple-helix is low, then the product is the denatured form of collagen, gelatin. Gelatin consists of only single chains that are not folded into in the native triple-helical structure. Also, while recombinant technology can be used easily for small scale production of laboratory samples, for commercial scale production there will be manufacturing issues that will need to be solved and process economics evaluated.8 Initial biotechnology developments focused on the production of full-length human collagens, including the heterotrimeric type I collagen and the homotrimeric type III collagen.7 Mammalian cell systems, such as HT1080 and HEK 293-EBNA cells, have been used, but yields are very low. The same is also true for production in an insect cell system, Baculovirus. Both systems, however, do have endogenous P4H activity (Table 1). Microorganisms provide alternative expression systems and are a natural choice because of their familiarity in the biotechnology industry.9 Unfortunately, the use of E. coli expression was initially not seen as practical, as there seemed to be difficulties in co-expressing all the necessary genes.7 On the other hand, yeast systems were considerably more promising, and several have been used successfully for the expression of full-length hydroxylated human collagens (Table 1).10-12 The advantage of the yeasts is they can be optimized to integrate and co-express 3 or 4 genes concurrently. The most successful application, which has provided commercial opportunities, has emerged from expression in P. pastoris (Table 1).11 Yeasts, as with other microorganisms typically require different codon preferences to those in mammalian systems for optimal expression. The development of rapid gene synthesis enables effective codon optimized sequences as well as specific variants of these sequences to be readily produced resulting in giving good yield.
228
Recombinant Bacterial Collagen: An Emerging System Collagens were seen historically as being associated with multicellular animal tissues. In more recent times the suggestion of collagen-like molecules in other species has emerged, with (Gly-Xaa-Yaa)n repeating sequences present in a few fungi,20 in viruses,21 and in phage.22 Most interesting, however, are the numerous collagen-like structures that are being identified in bacteria and in the emerging, large numbers of bacterial genomes. Thus, a gene with a collagen-like repeat, that is a virulence factor, was identified and characterized in the bacterium Streptococcus pyogenes.23 Subsequently, a second, structurally distinct, collagenlike gene was identified from the same species.24 Both were shown by biochemical and biophysical studies to have the characteristic collagen triple-helical structure.25 The surprise that emerged was that these collagens were both stable around mammalian body temperature, with melting temperatures of 36.4 °C and 37.6 °C, without the presence of any hydroxyproline.25 A decade ago, Rasmussen and colleagues reported an analysis of the available bacterial genome databases, searching for collagen-related structural motifs (CSM).26 The study included 137 eubacterial genomes and 15 archaebacterial genomes and looked for sequences with homology to (Gly-Pro-Pro) n where n was 7 or more. This identified 53 proteins in 25 bacterial
Bioengineered
Volume 5 Issue 4
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UNAM Ciudad Universitaria] at 03:01 05 January 2015
Figure 1. A transmission electron micrograph of the collagen fibrils in a sheep anterior cruciate ligament, showing numerous collagen fibrils, all of similar diameter. Bacterial collagens do not form such fibrillar structures. Bar = 0.25 µm.
Other approaches to improve commercial yields are also possible and include modification of the propeptide domains, enhanced integration, increased copy number of introduced genes, and reduced production temperature and oxygen enrichment to increase the P4H activity. Other systems have also emerged, based on transgenic organisms. For collagens, the mammary gland has been used for collagen production in mouse milk (Table 1).13 Another, more unusual system that is suited for fibrous proteins has been production of recombinant human type III procollagen in cocoons of transgenic Bombyx mori silkworms.14 Collagen production in transgenic plants has also been developed and seems to provide cost-effective, commercial outcomes. Initial reports produced unhydroxylated or poorly hydroxylated collagen from leaves15 and from seeds.16 Additional introduction of the mammalian P4H is necessary for a quality collagen product, even though plants contain a form of P4H. Most recently, heterotrimeric collagen has been produced with P4H also present, giving excellent protein yields, which can be commercialized.17 There may be further advantages of recombinant systems that possibly work only at a laboratory level. For example a range of constructs have been made for type II collagen, where specific “D-period” repeats, about 234 amino acids each, have been removed or substituted into the structure, allowing a better understanding of collagen structure and function.18 Another variation to this approach has been the design and production of small collagen fragments with embedded multiple repetitive domains (integrin) within the collagen chain.19
Table 1. Examples of recombinant systems for human collagen production * Production systems
Yield
Comments
HT1080
≤1 mg/L
Mainly type II collagen and variants
HEK293
≤80 mg/L
Used to produce collagen types V, VII, VIII, X, XVI
CHO
