A number of bacterial collagen-like proteins with Gly as every third residue and a high Pro content have been observed to form stable triple-helical structures despite the absence of hydroxyproline (Hyp). Here, the high yield cold-shock expression system is used to obtain purified recombinant collagen-like protein (V-CL) from Streptococcus pyogenes containing an N-terminal globular domain V followed by the collagen triple-helix domain CL and the modified construct with two tandem collagen domains V-CL-CL. Both constructs and their isolated collagenous domains form stable triple-helices characterized by very sharp thermal transitions at 35-37°C and by high values of calorimetric enthalpy. Procedures for the formation of collagen SLS crystallites lead to parallel arrays of in register V-CL-CL molecules, as well as centrosymmetric arrays of dimers joined at their globular domains. At neutral pH and high concentrations, the bacterial constructs all show a tendency towards aggregation. The isolated collagen domains, CL and CL-CL, form units of diameter 4-5 nm which bundle together and twist to make larger fibrillar structures. Thus, although this S. pyogenes collagen-like protein is a cell surface protein with no indication of participation in higher order structure, the triple-helix domain has the potential of forming fibrillar structures even in the absence of hydroxyproline. The formation of fibrils suggests bacterial collagen proteins may be useful for biomaterials and tissue engineering applications.

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not illicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37°C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

OBJECTIVE To compare efficacy and safety of dulaglutide at doses of 3.0 and 4.5 mg versus 1.5 mg in patients with type 2 diabetes inadequately controlled with metformin. RESEARCH DESIGN AND METHODS Patients were randomly assigned to once-weekly dulaglutide 1.5 mg, 3.0 mg, or 4.5 mg for 52 weeks. The primary objective was determining superiority of dulaglutide 3.0 mg and/or 4.5 mg over 1.5 mg in HbA 1c reduction at 36 weeks. Secondary superiority objectives included change in body weight. Two estimands addressed efficacy objectives: treatment regimen (regardless of treatment discontinuation or rescue medication) and efficacy (on treatment without rescue medication) in all randomly assigned patients. RESULTS Mean baseline HbA 1c and BMI in randomly assigned patients ( N = 1,842) was 8.6% (70 mmol/mol) and 34.2 kg/m 2 , respectively. At 36 weeks, dulaglutide 4.5 mg provided superior HbA 1c reductions compared with 1.5 mg (treatment-regimen estimand: −1.77 vs. −1.54% [−19.4 vs. −16.8 mmol/mol], estimated treatment difference [ETD] −0.24% (−2.6 mmol/mol), P < 0.001; efficacy estimand: −1.87 vs. −1.53% [−20.4 vs. −16.7 mmol/mol], ETD −0.34% (−3.7 mmol/mol), P < 0.001). Dulaglutide 3.0 mg was superior to 1.5 mg for reducing HbA 1c , using the efficacy estimand (ETD −0.17% [−1.9 mmol/mol]; P = 0.003) but not the treatment-regimen estimand (ETD −0.10% [−1.1 mmol/mol]; P = 0.096). Dulaglutide 4.5 mg was superior to 1.5 mg for weight loss at 36 weeks for both estimands (treatment regimen: −4.6 vs. −3.0 kg, ETD −1.6 kg, P < 0.001; efficacy: −4.7 vs. −3.1 kg, ETD −1.6 kg, P < 0.001). Common adverse events through 36 weeks included nausea (1.5 mg, 13.4%; 3 mg, 15.6%; 4.5 mg, 16.4%) and vomiting (1.5 mg, 5.6%; 3 mg, 8.3%; 4.5 mg, 9.3%). CONCLUSIONS In patients with type 2 diabetes inadequately controlled by metformin, escalation from dulaglutide 1.5 mg to 3.0 mg or 4.5 mg provided clinically relevant, dose-related reductions in HbA 1c and body weight with a similar safety profile.