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- Bandmann, Nina, 1971-
(författare)
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Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bacterium Escherichia coli (E. coli) is in many situations an ideal host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve sufficiently high product quantities. However, there are several factors that may limit this host’s ability to produce large amounts of heterologous proteins in a soluble and native form. For many applications a high purity of the recombinant protein is demanded, which implies a purification strategy where the product efficiently can be isolated from the complex milieu of host cell contaminants. In this thesis, different strategies based on both rational and combinatorial genetic engineering principles have been investigated, aiming at improving and facilitating recombinant E. coli protein production and purification. One objective was to improve the PEG/salt aqueous two-phase system (ATPS) purification process of the lipase cutinase, by increasing the selectivity of the protein for the system top-phase. Peptide tags, with varying properties, were designed and genetically fused to the C-terminal end of ZZ-cutinase. Greatly increased partitioning values were observed for purified protein variants fused to tryptophan containing peptide tags, particularly a (WP)4 peptide. The partitioning properties of the ZZ-cutinase-(WP)4 protein were also retained when added to the ATPS directly from an E. coli total cell disintegrate, emphasizing the applicability of this genetic engineering strategy for primary protein purification in ATPSs. Further on, a combinatorial library approach using phage display technology was investigated as a tool for identification of peptide tags capable of improving partitioning properties of ZZ-cutinase in an ATPS. Repeated ATPS-based partitioning-selection cycles of a large phagemid (pVIII) peptide library, resulted in isolation of phage particles preferentially decorated with peptides rich in tyrosine and proline residues. Both a peptide corresponding to a phage library derived peptide sequence as well as peptides designed based on information of amino acid appearance frequencies in later selection rounds, were shown to improve partitioning several-fold when genetically fused to the C-terminal end of ZZ-cutinase. From the two- to four–fold increased production yields observed for these fusion proteins compared to ZZ-cutinase-(WP)4, it was concluded that the selection system used allowed for selection of desired peptide properties related to both partitioning and E. coli protein production parameters. Bacterial protein production is affected by several different mRNA and protein sequence-related features. Attempts to address single parameters in this respect are difficult due to the inter-dependence of many features, for example between codon optimization and mRNA secondary structure effects. Two combinatorial expression vector libraries (ExLib1 and ExLib2) were constructed using a randomization strategy that potentially could lead to variations in many of these sequence-related features and which would allow a pragmatic search of vector variants showing positive net effects on the level of soluble protein production. ExLib1 was constructed to encode all possible synonymous codons of an eight amino acid N-terminal extension of protein Z, fused to the N-terminal of an enhanced green fluorescent reporter protein (EGFP). In ExLib2, the same eight positions were randomized using an (NNG/T) degeneracy code, which could lead to various effects on both the nucleotide and protein level, through the introduction of nucleotide sequences functional as e.g. alternative ribosome binding or translation initiation sites or as translated codons for an Nterminal extension of the target protein by a peptide sequence. Flow cytometric analyses and sorting of library cell cultures resulted in isolation of clones displaying several-fold increases in whole cell fluorescence compared to a reference clone. SDS-PAGE and western blot analyses verified that this was a result of increases (up to 24-fold) in soluble intracellular ZEGFP product protein content. Both position specific codon bias effects and the appearance of new ribosomal binding sites in the library sequences were concluded to have influenced the protein production. To explore the possibility of applying the same combinatorial library strategy for improving soluble intracellular production of heterologous proteins proven difficult to express in E. coli, three proteins with either bacterial (a transcriptional regulator (DntR)) or human (progesterone receptor ligand binding domain (PRLBD) and 11-β Hydroxysteroid dehydrogenase type I (11-β)) origin, were cloned into the ExLib2 library. Flow cytometric sorting of libraries resulted in isolation of DntR library clones showing increased soluble protein production levels and PR-LBD library clones with up to ten-fold increases in whole cell fluorescence, although the product under these conditions co-separated with the insoluble cell material.
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| 2. |
- Edwin, Aaron, 1983-
(författare)
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Structural and functional studies of the secreted metalloprotease PrtV from Vibrio cholerae
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.
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| 4. |
- Wallenhammar, Amélie
(författare)
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Protein modularity : Structure and interactions by NMR and SPR
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein degradation is a fundamental cornerstone of regulation of protein levels in the human body. For proper function, numerous molecular processes rely on fine-tuned communication through specific interactions between proteins. Tagging proteins by a small protein ubiquitin, called ubiquitination, is mediated by set of ubiquitin-conjugating and transferring enzymes with ubiquitin as well as substrates. This process enables specific selection of substrate modifications, which is directly coupled to biological activity.In this work, the E3 ligase enzyme TRIM21, a member of the TRIM (TRIpartite Motif) protein family, and its interactions are explored. First, the three-dimensional solution structure of the TRIM21 B-box2 domain solved by nuclear magnetic resonance (NMR) spectroscopy is presented. The structure reveals how different surfaces of the B-box motif are employed for various modular interactions. Using NMR titration experiments, an exposed interaction surface is identified, described as a novel interaction patch where the B-box2 is likely to bind the RING domain. Taken together, this work establishes an extended understanding of the structural role of the B-box2 domain, and how this domain is related to flanking domains in multimodular TRIM proteins.Our crystal structure of TRIM21-UBE2E1 complex reveals the specific E2 recognition, which is conserved in other TRIMs. Additionally, solution scattering provides further information on the complexes and also confirms that TRIM21 RING1-91 is predominantly dimer. We also understand how the acceptor ubiquitin can be transferred to Lysine residue of TRIM211-91.Another highly regulated system is the RNA polymerase-II transcription, which is the first step for all protein synthesis. TATA-box Binding Protein (TBP) is an essential subunit for initiating the gene transcription, which is regulated by number of other transcription factors. Among these, the oncoprotein c-Myc regulates the transcription by directly mediating its interaction with TBP. The interplay of these interaction and competing binding of TBP and c-Myc is studied in this thesis.Conclusively, this thesis highlights TRIM molecular recognition and its interface with E2, and also provides the first glimpse of interaction between TBP and c-Myc.
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