High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.Pubblicat

Modified polyethylene glycols and their supramolecular complexes with biologically active macromolecules

The present invention in a first aspect relates to a polymer based on modified polyethylene glycol (PEG) of formula CH3-(OCH2CH2)n-O-X-A, wherein (OCH2CH2)n- is a polyoxyethylene chain wherein n varies from 100 to 5000, X is a saturated or unsaturated C1C20 linear alkyl group or a saturated or unsaturated C3-C5 branched alkyl group having at least one end functionalised with a hydroxyl, amino, carboxylic or thiol group, and A is a functional lipophilic group. In another aspect, the invention relates to supramolecular complexes of the polymers of polyethylene glycol (PEG) that is modified with a biologically active macromolecule or a biopharmaceutical and their applications in therapy. (FR)L'invention concerne, selon un premier aspect, un polym\ue8re \ue0 base de poly\ue9thyl\ue8ne glycol modifi\ue9 (PEG) de formule CH3-(OCH2CH2)n-O-X-A, dans laquelle (OCH2CH2)n- est une cha\ueene polyoxy\ue9thyl\ue8ne dans laquelle n varie de 100 \ue0 5000; X est un groupe alkyle C1C20 lin\ue9aire satur\ue9 ou insatur\ue9, ou un groupe alkyle C3-C5 ramifi\ue9 satur\ue9 ou insatur\ue9 dont au moins une extr\ue9mit\ue9 est fonctionnalis\ue9e par un groupe hydroxyle, amino, carboxylique ou thiol; et A est un groupe lipophile fonctionnel. Dans un autre aspect, l'invention concerne des complexes supramol\ue9culaires des polym\ue8res de poly\ue9thyl\ue8ne glycol (PEG) modifi\ue9s avec une macromol\ue9cule biologiquement active ou un produit biopharmaceutique, et leurs applications th\ue9rapeutiques

Preclinical characterization of eleven new Cys-PEGylated hGH mutants

We synthesized and tested for biological activity eleven new PEGylated hGH derivatives. To this aim we used different strategies. A first group of PEGylable Cys derivatives was prepared by mutating into Cys single specific aminoacid residues located either in the connecting loop between helices 1 and 2 (Ile36 and Phe44) or in the connecting loop between helices 3 and 4 (Ile138 and Phe146). A second group of mutants was synthetized by inserting new Cys close to the aforementioned positions. Other PEGylable mutants were prepared by inserting a Gly4–Cys chain either at the N- or at the C-terminus of the hGH molecule. Finally, a Cys residue of the second hGH disulfide bond (Cys182–Cys189) was made available for PEGylation by mutating its companion Cys to open up the disulfide bridge. All these mutants were tested for their ability to affect Nb2 cell proliferation in vitro and showed different effects. Two mutations (Phe44Cys and +Cys47) severely impaired and two (Ile138Cys and Phe146Cys) increased hGH bioactivity. The mutation Cys189Ser was functionally silent whereas the remaining mutations caused a 15–50% decrease in activity. 20 kDa-pegylation caused a dramatic decrease in bioactivity in all mutants. The r-hGH-(Ile138Cys)–Cys138–PEG derivative showed the highest activity (15% of wild-type unpegylated hGH) and was selected for pharmacockinetic studies in vivo. It showed a fivefold longer half-life and was significantly more effective than wild-type hGH in causing weight gain when given subcutaneously twice a week to hypophysectomized rats. In conclusion, even when it is directed to residues supposed to have a marginal role in the activity of this hormone, 20 kDa pegylation has detrimental effect on hGH bioactivity. These effects may be counterbalanced by the increase in half-life as it happens in pegylated Ile138Cys–hGH that could represent a promising new long-acting hGH derivative. Focal points: • 'Bedside': Current therapy of GH deficiency still has the important limitation of being delivered by daily subcutaneous injections and this reduces the compliance of the small pediatric patients. There is, therefore interest in developing long-acting GH derivative such as the new ones that we present here. • 'Benchside': By investigating the effect of Cys substitutions at aminoacidic positions that had not been investigated in previous studies, we obtained evidence that Ile138 and Phe146 have a role in GH bioactivity in vitro. • 'Industry': The mono-pegylated hGH mutant described in this work represents a promising candidate for human clinical pharmacology studies as long-acting derivative of h-GH.• 'Community': By decreasing the frequency of GH injections, long acting GH could decrease the costs of GH treatment and increase medication adherence

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen

Filgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34(+)) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E. coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen (R), a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen (R) in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics. Methods: Cell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen (R) to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence. Results: The results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen (R). Conclusions: BK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen (R) and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients

Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation.

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values