Hans Peter Sørensen

Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags.

Initiation of Protein Synthesis in Bacteria

SUMMARY Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process. Subsequent sections describe the structure, function, and interactions of the mRNA, the initiator tRNA, and the initiation factors IF1, IF2, and IF3. Finally, we provide an overview of mechanisms of regulation of the translation initiation event. Translation occurs on ribonucleoprotein complexes called ribosomes. The ribosome is composed of a large subunit and a small subunit that hold the activities of peptidyltransfer and decode the triplet code of the mRNA, respectively. Translation initiation is promoted by IF1, IF2, and IF3, which mediate base pairing of the initiator tRNA anticodon to the mRNA initiation codon located in the ribosomal P-site. The mechanism of translation initiation differs for canonical and leaderless mRNAs, since the latter is dependent on the relative level of the initiation factors. Regulation of translation occurs primarily in the initiation phase. Secondary structures at the mRNA ribosomal binding site (RBS) inhibit translation initiation. The accessibility of the RBS is regulated by temperature and binding of small metabolites, proteins, or antisense RNAs. The future challenge is to obtain atomic-resolution structures of complete initiation complexes in order to understand the mechanism of translation initiation in molecular detail.

Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck

Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E. coli often results in truncated protein forms. In many cases, this is a consequence of abundant codons in heterologous genes, which are decoded by rare tRNAs in E. coli-a combination that can be responsible for translational stalling and termination during protein biosynthesis. Other complications may originate from potential initiation codons and ribosomal binding sites present inside the open reading frame of the target gene or from other less well defined phenomena such as mRNA instability. Separation of full-length protein from truncated forms is a serious chromatographic problem that can be solved in the expression step. We have investigated the heterologous expression and purification of two translation initiation factors from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus. Expression in E. coli was optimised to avoid truncated forms completely by complementation with the plasmids pSJS1244, pRIG, pCODON+ and pLysSR.A.R.E harbouring and expressing genes encoding rare tRNAs corresponding to the codons AGA, AGG, AUA, CUA, GGA, AAG and CCC. Two expression strains, C41(DE3) and C43(DE3) were found highly advantageous when combined with rare tRNA encoding plasmids as compared to BL21(DE3). We have also investigated the effects of site directed mutagenesis on rare lysine encoding AAG doublets as well as two methionine residues preceded by potential ribosomal binding sites. The expression approach presented here has enabled us to purify gram quantities of full-length protein by one step of ion-exchange chromatography and is generally applicable to many other heterologously expressed thermostable proteins.

Heparan Sulfate Regulates ADAM12 through a Molecular Switch Mechanism

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.

Dialysis strategies for protein refolding: preparative streptavidin production

We have investigated different dialysis strategies for the refolding of recombinant streptavidin, and present a novel dialysis setup featuring gradual dilution dialysis and continuous protein feeding into a dialysis sack. A denaturing dialysis buffer is exchanged gradually by dilution with refolding buffer and it is demonstrated that the refolding yield can be increased from 45 to 75% by lowering the dilution rate. In addition, continuous feeding of protein to the dialysis sack increases the yield by 5 to 10%. The principle of gradual dilution dialysis is amenable to stringent regulation and we suggest it to be applied for other insoluble protein targets.

Remarkable Conservation of Translation Initiation Factors: IF1/eIF1A and IF2/eIF5B are Universally Distributed Phylogenetic Markers

Initiation of protein biosynthesis is an essential process occurring in cells throughout the three phylogenetic domains, Bacteria, Archaea, and Eucarya. IF1/eIF1A and IF2/eIF5B, two conserved translation initiation factors are involved in this important step of protein biosynthesis. The essentiality, universal distribution, conservation, and interspecies functional homology of both factors are a unique combination of properties ideal for molecular phylogenetic studies as demonstrated by the extensively compared SSU rRNAs. Here, we assess the use of IF1/eIF1A and IF2/eIF5B in universal and partial phylogenetic studies by comparison of sequence information from species within all three phylogenetic domains and among closely related strains of Haemophilus parainfluenzae . We conclude that the amino acid sequence of IF1/eIF1A‐IF2/eIF5B is a universal phylogenetic marker and that the nucleotide sequence of the IF2/eIF5B G‐domain is more credible than SSU rRNA for the construction of partial phylogenies among closely related species and strains. Because of these two application levels, IF1/eIF1AIF2/eIF5B is a phylogenetic “dual level” marker.

The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms.

Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

Macromolecular Mimicry in Translation Initiation: A Model for the Initiation Factor IF2 on the Ribosome

Protein biosynthesis in bacteria is controlled by a number of translation factors. Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A‐site‐bound tRNA. This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2. Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex.

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.