Waltraud Kaar

N(pro) fusion technology to produce proteins with authentic N termini in E. coli.

We describe a prokaryotic expression system using the autoproteolytic function of Npro from classical swine fever virus. Proteins or peptides expressed as Npro fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus. A tailor-made Npro mutant called EDDIE, with increased in vitro and decreased in vivo cleavage rates, has enabled us to express proinsulin, domain-D of staphylococcal protein A, hepcidin, interferon-α1, keratin-associated protein 10-4, green fluorescent protein, inhibitorial peptide of senescence-evasion-factor, monocyte chemoattractant protein-1 and toxic gyrase inhibitor, among others. This Npro expression system can be used as a generic tool for the high-level production of recombinant toxic peptides and proteins (up to 12 g/l) in Escherichia coli without the need for chemical or enzymatic removal of the fusion tag.

Refolding of Npro fusion proteins

The autoprotease Npro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C‐terminus releasing the target protein with its authentic N‐terminus. Npro wild‐type and its mutant EDDIE, respectively, were fused N‐terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence‐evasion‐factor, and the short 16 amino acid peptide pep6His. In comparison with the Npro wild‐type, the tailored mutant EDDIE displayed an increased rate constant for refolding and cleavage from 1.3 × 10−4 s−1 to 3.5 × 10−4 s−1, and allowed a 15‐fold higher protein concentration of 1.1 mg/mL when studying pep6His as a fusion partner. For green fluorescent protein, the rate constant was increased from 2.4 × 10−5 s−1 to 1.1 × 10−4 s−1 when fused to EDDIE. When fused to small target peptides, refolding and cleavage yields were independent of initial protein concentration, even at high concentrations of 3.9 mg/mL, although cleavage rates were strongly influenced by the fusion partner. This behavior differed from conventional 1st order refolding kinetics, where yield strongly depends on initial protein concentration due to an aggregation reaction of higher order. Refolding and cleavage of EDDIE fusion proteins follow a monomolecular reaction for the autoproteolytic cleavage over a wide concentration range. At high protein concentrations, deviations from the model assumptions were observed and thus smaller rate constants were required to approximate the data. Biotechnol. Bioeng. 2009; 104: 774–784

Expression and purification of a nanostructure-forming peptide

Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P(11)-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.

Microbial Bio-Production of a Recombinant Stimuli-Responsive Biosurfactant

Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum‐derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single‐step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent‐, acid‐, and heat‐mediated precipitation of impurities were successfully applied as alternatives to post‐cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio‐produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam‐stabilizing and foam‐destabilizing states. Biotechnol. Bioeng. 2009;102: 176–187.

Npro Autoprotease Fusion Technology: Development, Characteristics, and Influential Factors

Npro fusion technology enables the overexpression of peptides/proteins fused to an autoprotease and deposited as inclusion bodies in E. coli. Under kosmotropic conditions, the autoprotease cuts itself at the C terminus, releasing the target with an authentic N terminus. A tailor-made Npro mutant, EDDIE, has overcome problems with low solubility and cleavage rate of wild-type Npro. Codon optimization avoids truncation and prolongation of the fusion proteins. The examples presented demonstrate important factors (first amino acid of target, type of chaotrope) to optimize for raising the rate constant and yield of the autoproteolytic reaction, thus increasing protein production productivity up to 10-fold.

The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly

One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography‐free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide‐containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self‐assembling β‐sheet forming peptide P11‐2 in fusion to thioredoxin. Homogenate was heat treated (55°C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P11‐2 having a native N‐terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation‐purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P11‐2 from partitioning to the precipitate. This process structure gave recombinant P11‐2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer‐exchange of the 97% pure product by bind‐elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self‐assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography‐free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost‐sensitive materials applications for peptide‐based materials. Biotechnol. Bioeng. 2009; 104: 973–985.

Peptide affinity chromatography media that bind Npro fusion proteins under chaotropic conditions

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N(pro) fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N(pro) from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7-15 mg protein mL(-1) of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.