Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation.

The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

Unnatural proteins for the control of surface forces.

We introduce a new method for the stabilization of colloidal particles via the synthesis and adsorption of unnatural proteins. Biosynthesis of protein-based polymers offers the advantages of preparation of complex sequences through control of the primary sequence, monodisperse polymers, ease of combinatorial search for anchor blocks, environmentally friendly synthesis, use of water as the solvent, and incorporation of a palette of known natural proteins. We have synthesized an unnatural protein with the sequence thioredoxin-Pro(39)Glu(10) for modification of the forces between alumina particles. The polyglutamate sequence, Glu(10), is anionic (pH > 3) and is designed to anchor the protein to positively charged solids, e.g. alumina in water (pH < 9). The polyproline sequence, Pro(39), is neutral. The thioredoxin is a recombinant form of the natural globular protein with a histidine patch (His-patch-thioredoxin) and is zwitterionic. The combined thioredoxin-Pro(39) sequence is hydrophilic with pI approximately 6.3. This block is designed to remain in solution, thereby providing a steric barrier to the approach of two particles in a range of salt and pH conditions. Ellipsometry experiments show that thioredoxin-Pro(39)Glu(10) does adsorb to alumina. Force measurements with the atomic force microscopy (AFM) colloid probe technique show that adsorption of thioredoxin-Pro(39)Glu(10) leads to repulsive forces that decay exponentially with the separation between the surfaces and are independent of salt concentration in the range 0.001-0.1 M KNO(3). This demonstrates that the repulsive forces are not electrostatic. We hypothesize that the repulsion is due to confinement and loss of solvent for the adsorbed polymer; the forces are similar to those expected for a polymer brush. Force measurements between thioredoxin-coated alumina surfaces also show a repulsive force, but the force has a decay length that is consistent with electrostatic double-layer forces: the thioredoxin has not neutralized the surface charge of the underlying alumina. Our results point to interesting future experiments where recombinant DNA technology could be used to synthesize fusion proteins containing useful natural proteins and an anchor. This may allow preparation, via single-step aqueous self-assembly, of anchored proteins that maintain their natural structure. Our technique is not limited to homopolymer blocks; more complex primary sequences can be used.

A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L. plants.

By analysing two-dimensional patterns of chloroplastic proteins from Solanum tuberosum, the authors observed the accumulation of a 32-kDa polypeptide in the stroma of plants subjected to water deficit. N-terminus and internal peptides of the protein, named CDSP 32 for chloroplastic drought-induced stress protein, showed no obvious homology with known sequences. Using a serum raised against the protein N-terminus, a cDNA encoding CDSP 32 was cloned by screening an expression library. The deduced mature CDSP 32 protein is 243 amino acids long and displays typical features of thioredoxins in the C-terminal region (122 residues). In particular, CDSP 32 contains a CGPC motif corresponding to a thioredoxin active site and a number of amino acids conferring thioredoxin-type structure. The CDSP 32 C-terminal region was expressed as a fusion protein in Escherichia coli and was shown to possess thioredoxin activity based on reduction assay of insulin disulfide bridges. RNA blot analysis showed that CDSP 32 transcript does not accumulate upon mild water deficit conditions corresponding to leaf relative water contents (RWC) around 85%, but high levels of CDSP 32 transcripts were observed for more severe stress conditions (RWC around 70%). In vivo labelling and immunoprecipitation revealed a substantial increase in CDSP 32 synthesis upon similar stress conditions. Rewatering of wilted plants caused decreases in both transcript and protein abundances. In tomato wild-type plants and ABA-deficient mutants, a similar accumulation of a CDSP 32-related transcript was observed upon water deficit, most likely indicating no requirement for ABA in the regulation of CDSP 32 synthesis. Based on these results, it is proposed that CDSP 32 plays a role in preservation of the thiol: disulfide redox potential of chloroplastic proteins during water deficit.