Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers

Han, Runlin; Zwiefka, Antoni; Caswell, Clayton C.; Xu, Yi; Keene, Douglas R.; Lukomska, Ewa; Zhao, Zhihong; Höök, Magnus; Łukomski, Sławomir

doi:10.1007/s00253-006-0387-5

Cited by 76 publications

(111 citation statements)

References 35 publications

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“…While some triplets identified in human collagens that are part of the integrin binding sites, such as GFO, GMO, and GEN, are never found within the Scl-CL regions, other triplets may occur frequently, including GER (3.20%), GEK (7.08%), and GLP (4.89%) (18). Interestingly, the RGD cryptic motif is common in the Scl2-CL regions 3 and is also found in recently reported SclZ protein (38).…”

Section: Discussionmentioning

confidence: 99%

“…2). Our earlier EM studies revealed that rScls share a common "lollipop-like" two-domain organization composed of the globular head formed by the V region and the lollipop stalk made by the fibrous CL region (17,18). Binding events between the r␣ 2 I domain and the CL region of P222 were observed in electron micrographs, and the location of bound r␣ 2 I appeared to be the same in each case.…”

Section: Glpger Sequence In Rscl Is Sufficient For Integrin Binding-mentioning

confidence: 98%

“…E. coli strains harboring plasmid constructs were routinely grown in Luria-Bertani (LB) liquid medium (BD Biosciences) supplemented with ampicillin (100 g/ml). Recombinant Scl (rScl) proteins (Table 1) were produced using the Strep-tag II cloning, expression, and purification system (IBA-GmbH, Goettingen, Germany), as described previously (17,18).…”

Section: Methodsmentioning

confidence: 99%

“…Similar to mammalian collagens, the collagen-like (CL) regions of Scl proteins adopt stable triple helices with midpoint melting temperatures of about 37°C (17,18). Because of the lack of prolyl hydroxylase in GAS, Scl proteins are devoid of O residues, which in mammalian collagens are regarded as essential for triple helix stabilization.…”

mentioning

confidence: 99%

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Identification of the First Prokaryotic Collagen Sequence Motif That Mediates Binding to Human Collagen Receptors, Integrins α2β1 and α11β1

Caswell

Barczyk

Keene

et al. 2008

Journal of Biological Chemistry

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Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin ␣ 2 ␤ 1 . The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the ␣ 2 ␤ 1 and the ␣ 11 ␤ 1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant ␣ 2 -integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-␣ 2 ؉ myoblasts expressing the ␣ 2 ␤ 1 integrin as the sole collagen receptor. The C2C12-␣ 11 ؉ myoblasts expressing the ␣ 11 ␤ 1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-␣ 11 ؉ cells attached to rScl1 more efficiently than C2C12-␣ 2 ؉ cells, suggesting that the ␣ 11 ␤ 1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria.The integrins are a family of ␣␤ heterodimeric cell surface receptors found in metazoans that bind proteins of the extracellular matrix (ECM) and function in diverse cellular processes, including cell adhesion, spreading, and migration. There are currently 18 ␣ and 8 ␤ known subunits that combine to form 24 distinct heterodimers with various ligand binding specificities (1). One group of integrins binds several collagen types with varying affinities and includes the integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 (2). The ␣ subunit of these integrins contains an inserted (I) 2 domain, and the ␣I domain confers binding specificity to the integrin heterodimer. Over the past decade much emphasis has been directed toward defining the parameters of collagen recognition by these integrins, as well as the specific amino acid sequences in collagens that are required for binding.The GFOGER (where O is hydroxyproline) sequence has been identified in some collagens as a major binding motif for the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins (3, 4). However, not all collagen types harbor the GFOGER motif. Using synthetic collagen peptides, additional sequences with the base sequence GXOGER were proposed to bind the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins with lower affinity as compared with the GFOGER sequence (5, 6). The hydroxylated proline residue of GXOGER is required for binding by the ␣ 1 ␤ 1 integrin but not by the ␣ 2 ␤ 1 integrin (7). Furthermore, the E residue of the GER triplet is essential for integ...

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Section: Discussionmentioning

confidence: 99%

Section: Glpger Sequence In Rscl Is Sufficient For Integrin Binding-mentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of the First Prokaryotic Collagen Sequence Motif That Mediates Binding to Human Collagen Receptors, Integrins α2β1 and α11β1

Caswell

Barczyk

Keene

et al. 2008

Journal of Biological Chemistry

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“…30 The identification of this new group of collagens provides potential for development of new, recombinant biomedical materials. 31 Several have now been studied after expression in E. coli. 28 They do not contain any hydroxyproline, and even though some native bacterial structures are glycosylated, this is not the case with the recombinant products.…”

Section: Recombinant Bacterial Collagen: An Emerging Systemmentioning

confidence: 99%

Bioengineered collagens

2014

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Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Young

Britton

Robinson

2012

Biotechnology Journal

410

267

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Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

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Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers

Cited by 76 publications

References 35 publications

Identification of the First Prokaryotic Collagen Sequence Motif That Mediates Binding to Human Collagen Receptors, Integrins α2β1 and α11β1

Identification of the First Prokaryotic Collagen Sequence Motif That Mediates Binding to Human Collagen Receptors, Integrins α2β1 and α11β1

Bioengineered collagens

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

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