A fluorescence-based array screen for transglutaminase substrates.

Transglutaminases (EC 2.3.2.

Transglutamination allows production and characterization of native-sized ELPylated spider silk proteins from transgenic plants.

In the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant-based production technology especially for protein-based products, for technical use as fibres or biodegradable plastics and also for medical applications. High-performance fibres from recombinant spider silks are, therefore, a prominent example.

Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag.

In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material.

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis.

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis.

Enzymatically catalyzed conjugation of a biodegradable polymer to proteins and small molecules using microbial transglutaminase.

Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is widely used in biomedicine as a plasma volume expander. Due to its favorable properties, HES is currently being investigated at the industrial and academic levels as a biodegradable polymer substitute for polyethylene glycol. To date, only chemical methods have been suggested for HESylation; unfortunately, however, these may have negative effects on protein stability.

Enzymatically catalyzed HES conjugation using microbial transglutaminase: Proof of feasibility.

Polymer-drug and polymer-protein conjugates are promising candidates for the delivery of therapeutic agents. PEGylation, using poly(ethylene glycol) for the conjugation, is now the gold standard in this field, and some PEGylated proteins have successfully reached the market. Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is currently being investigated as a substitute for PEG.

Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants.

Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or the posttranslational modification of (therapeutic) proteins, was optimized by random mutagenesis for the first time. A screening method was developed which, in addition to state-of-the-art procedures, includes a proteolytic activation step of the expressed soluble pro-enzyme. The library of 5,500 clones was screened for variants with increased thermostability and heat-sensitivity, respectively.

Cloning, expression, purification, and characterization of a designer protein with repetitive sequences.

An artificial protein containing alternating hydrophilic-hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy.