Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.

Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha.

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.

Alkaline phosphatase from the Antarctic strain TAB5. Properties and psychrophilic adaptations.

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology-based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations. The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity. The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.

Preparation of high purity alkaline phosphatase from calf intestine using dye-ligand chromatography.

For the effective application of alkaline phosphatase from calf intestine in Molecular Biology research highly purified enzyme and free from contaminating DNases, DNA nicking, ribonuclease and phosphodiesterase activities is required. We now report the use of a two-step procedure which involves chromatography on a Mimetic Blue AP-Agarose, a commercially available adsorbent and Heparin Sepharose to purify calf intestinal alkaline phosphatase from a crude commercial preparation to homogeneity. Purified enzyme preparations were free from contaminating DNases, DNA nicking, ribonuclease and phosphodiesterase activities and exhibited a specific activity (3.800 units/mg) which is one of the highest reported among the existing high purity commercial preparations. It is therefore concluded that the reported purification protocol can be used routinely to prepare high purity alkaline phosphatase suitable for use in Molecular Biology research.

Sequence-specific DNA affinity chromatography: application of a group-specific adsorbent for the isolation of restriction endonucleases.

The use of sequence-specific DNA affinity adsorbents for the isolation of restriction endonucleases EcoRI and SphI to near homogeneity has been reported. However, the high cost of these adsorbents is a limiting factor for their wider application. This paper reports the application of sequence-specific DNA affinity ligands containing recognition sequences for 34 restriction endonucleases as group-specific ligands in the isolation of restriction endonucleases. Crude samples of six restriction endonucleases, namely BshFI, BamHI, SmaI, SacII, PvuII and SalI, were shown to bind to these adsorbents and could be eluted at different KCl concentrations. High purification factors and recoveries were obtained. Restriction endonuclease BshFI, an isoschizomer of HaeIII, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose chromatography and oligonucleotide-ligand affinity chromatography. The enzyme exists as a monomer with an apparent relative molecular mass of 34,000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion chromatography.