Codon Preference Optimization Increases Heterologous PEDF Expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes , respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ∼3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ∼4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ∼3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at −3 to +50 of the Trx site of both fusion genes were −3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo

Asymmetric total synthesis of erythromycin. 3. Total synthesis of erythromycin

In the preceding paper' we described the preparation of the key lactone intermediate la in optically active form. In this paper we report the synthesis of erythromycin (2) from la. In essence,this transformation involves the glycosidation of a suitable derivative of la with L-cladinose and D-desosamine and the generation of the C-9 ketone functionality

RE-OPEN: Randomised trial of biosimilar TNK versus TPA during endovascular therapy for acute ischaemic stroke due to large vessel occlusions

Rationale Rapid and timely treatment with intravenous thrombolysis and endovascular treatment (EVT) in patients with acute ischaemic stroke (AIS) and large vessel occlusion (LVO) significantly improves patient outcomes. Bridging therapy is the current standard of care in these patients. However, an incompletely answered question is whether one thrombolytic agent is better than another during bridging therapy.Aim The current study aims to understand if one thrombolytic agent is superior to the other during bridging therapy in the treatment of AIS and LVO.Sample size estimates Using 80% power and an alpha error of 5 %, presuming a 10% drop out rate, a total of 372 patients will be recruited for the study.Methods and design This study is a prospective, randomised, multicentre, open-label trial with blinded outcome analysis design.Study outcomes The primary outcomes include proportion of patients who will be independent at 3 months (modified Rankin score (mRS) ≤2 as good outcome) and proportion of patients who achieve recanalisation modified thrombolysis in cerebral infarction grade 2b/3 at first angiography run at the end of EVT. Secondary outcomes include proportion of patients with early neurological improvement, rate of symptomatic intracerebral haemorrhage (ICH), rate of any ICH, rate of any systemic major or minor bleeding and duration of hospital stay. Safety outcomes include any intracranial bleeding or symptomatic ICH.Discussion This trial is envisioned to confirm the theoretical advantages and increase the strength and quality of evidence for use of tenecteplase (TNK) in practice. Also, it will help to generate data on the efficacy and safety of biosimilar TNK.Trial registration number CTRI/2022/01/039473