Understanding heterologous protein overproduction under the T7 promoter - A practical exercise

Because various genome projects have been advanced many genes are known, and large amounts of proteins are required to elucidate their function. Most biomolecular research laboratories have a need to overexpress a certain gene, or a part of it, in eukaryotic or prokaryotic expression systems. It is therefore important for young students to become familiar with the technology of heterologous gene expression systems. Gene expression in eukaryotic cells is rather complicated and costly and is therefore not ideally suited to exercises for students. The goal of this paper is to describe an experimental example of a well known and broadly used prokaryotic system, the pET system, that works under the strong T7 promoter. The clones described in this paper are suitable for the practical exercise and are available upon request

Cloning, overproduction, purification and crystallization of the DNA binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima

The humar gene encoding for the histone-like DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima was efficiently overexpressed in Escherichia coli under the T7 promoter. The HU protein was purified using SP-Sepharose ion-exchange and heparin-affinity chromatography and was successfully crystallized in ammonium sulfate. The crystals were grown in the tetragonal form in space group P4(3) or P4(1) and have unit-cell dimensions a = b = 46.12, c = 77.56 Angstrom, a = beta = gamma = 90 degrees. The crystals diffract X-rays to 1.6 Angstrom resolution using synchrotron radiation and are suitable for determination of the HU structure at high resolution

Isolation, cloning, and overexpression of a chitinase gene fragment from the hyperthermophilic archaeon Thermococcus chitonophagus: semi-denaturing purification of the recombinant peptide and investigation of its relation with other chitinases

A 189-bp sequence was isolated from the hyperthermophilic archaeon Thermococcus chitonophagus and was found to present strong homology with a large number of chitinase genes from a variety of organisms and particularly with the chitinaseA gene from Pyrococcus kodakaraensis (Pk-chiA). This fragment was subcloned to an expression vector and overexpressed in Escherichia coli. The E coli BLR21(DE3)pLysS transformant, harbouring the gene on the pET-31b plasmid vector, was found to overproduce the target protein at high levels. The 63 aminoacid-long peptide was efficiently purified to homogeneity, with a one-step, semi-denaturing affinity chromatography, on a metal chelation resin and was used for the production of a specific, polyclonal antibody from rabbits. The produced antibody was demonstrated to display strong and specific affinity for the chitinase A from Serratia marcescens (Sm-chiA), as well as the membrane-bound chitinase70 from Thermococcus chitonophagus (Tc-Chi70). The strong sequence homology, in combination with the demonstrated specific immunochemical affinity, indicates that the isolated peptide is part of a chitinolytic enzyme of T chitonophagus. In particular, it could belong to the membrane-bound chi70, or to a distinct chitinase, coded by a different gene, or even by the same gene, following post-transcriptional or post-translational modifications. (C) 2004 Elsevier Inc. All rights reserved

Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis

A gene encoding a highly thermostable extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus was identified, The gene was cloned, sequenced, and expressed in Escherichia coli and Bacillus subtilis. The gene is 1383 base pairs long and encodes a protein of 461 amino acids, The open reading frame of the gene was Verified by microsequencing of the recombinant purified enzyme. The deduced amino acid sequence is 25 amino acids longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the membrane. The recombinant alpha-amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to 100 degrees C. alpha-Amylase shows sequence homology to the other known alpha-amylases and belongs to family 13 of glycosyl hydrolases. This extracellular alpha-amylase is not homologous to the subcellular alpha-amylase previously isolated from the same organism

Structure and dynamics of the DNA-binding protein HU of B-stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy

The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a 90-residue homodimer that binds nonspecifically to B DNA. Although the structure of the HUBst:DNA complex is not known, the proposed DNA-binding surface consists of extended arms that project from an alpha-helical platform. Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface. Raman conformation markers show that the DNA-binding arms of the dimer contain beta-stranded structure in excess (eight +/- two residues per subunit) of that reported previously. Important among side-chain markers are Met (701 cm(-1)), Ala (908 cm(-1)), Arg (1082 cm(-1)), and Pro (1457 cm(-1)). The Ala marker undergoes a substantial shift (908 –> 893 cm(-1)) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines. A large subset of alanines (67%) in the a-helical core exhibits robust resistance to exchange. A quantitative study of NH –> ND exchange exploiting newly identified amide II’ markers of helical (1440 cm(-1)) and nonhelical (1472 cm(-1)) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins

De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin

The purification scheme of chitinase A (ChiA) from S. marcescens has been extensively revised. The pure enzyme crystallizes readily under new crystallization conditions. The ChiA crystal structure has been refined to 1.55 Angstrom resolution and the crystal structure of ChiA co-crystallized with the inhibitor allosamidin has been refined to 1.9 Angstrom resolution. Allosamidin is located in the deep active-site tunnel of ChiA and interacts with three important residues: Glu315, the proton donor of the catalysis, Asp313, which adopts two conformations in the native structure but is oriented towards Glu315 in the inhibitor complex, and Tyr390, which lies opposite Glu315 in the active-site tunnel

Inhibition of two family 18 chitinases by various allosamidin derivatives

The inhibitory activities of several allosamidin derivatives on two family 18 chitinases, an insect enzyme from the epithelial cell line from Chironomus tentans, and a bacterial enzyme, chitinase A from Serratia marcescens, were evaluated. The following structural requirements are necessary for inhibition of the Chironomus enzyme: 1. One N-acetylallosamine residue can be omitted without impairment of enzyme inhibition. 2. At least one N-acetylallosamine sugar must be present. 3. Glucosamine can replace the allosamine moiety without a negative effect on the inhibitory activity. 4. The spatial arrangement of the allosamizoline moiety is important for inhibition. 5. If one sugar is omitted and the arrangement of the cyclitol residue is changed, the inhibitory effect is diminished further. For purified chitinase A from Serratia marcescens the arrangement of the aglycone moiety is equally important, but recognition of the sugar is different: 1. Omission of one allosamine residue decreases the inhibitory activity considerably. 2. Inhibition is improved if the remaining N-acetylallosamine is replaced by the epimer N-acetylglucosamine. Only endochitinase activity is affected, since chitin formation (up to 10(-4) M) and N-acetylglucosaminidase activity (up to 10(-3) M) are not impaired, at least in Chironomus cells. (C) 1998 SCI

Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation

The stereochemistry of the reaction catalysed by Serratia marcescens chitobiase was determined by HPLC separation of the anomers of N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc). In the early stages of the reaction, the beta-anomer was found to prevail, whereas the alpha-anomer dominated at mutarotation equilibrium. This established that chitobiase hydrolyses glycosidic bonds with overall retention of the anomeric configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was competitively inhibited by a series of chito-oligosaccharides (degree of polymerization 2-5) that were selectively de-N-acetylated at their non-reducing end. The results are in accord with the participation of the acetamido group at C-2 of the substrate in the catalytic mechanism of chitobiase and related enzymes

High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis

Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves beta -1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 Angstrom shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa- N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical “chair” to “boat” conformational change on the sugar residue bound to the - 1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of - 1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed “substrate assisted” catalysis

Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant

We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 mum of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomeric one. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding, whereas the quite small value for the unfolding heat capacity change (3.7 kJ.K-1.mol(-1)) stabilizes the protein within a broad temperature range, as shown by the stability curves (Gibbs energy functions vs. temperature), even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH 4.00 and 3.75, but becomes considerably less so at pH 3.50 and below, to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25 degreesC for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13