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On bacterial formats in protein library technology
KTH, School of Biotechnology (BIO), Molecular Biotechnology. (Molecular Biotechnology)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , xii,102 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009: 12
Keyword [en]
Affibody molecule, β-lactamase, combinatorial library, green fluorescent protein (GFP), protein engineering, protein fragment complementation assay (PCA), selection, TEVprotease, TNF-α
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:kth:diva-10993ISBN: 978-91-7415-400-9 (print)OAI: oai:DiVA.org:kth-10993DiVA: diva2:233785
Public defence
2009-09-25, AlbaNova Room FA 32, Roslagstullsbacken 21, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
QC 20100729Available from: 2009-09-08 Created: 2009-09-02 Last updated: 2011-12-08Bibliographically approved
List of papers
1. Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay
Open this publication in new window or tab >>Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay
2011 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 1, e16136- p.Article in journal (Refereed) Published
Abstract [en]

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P19, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-31599 (URN)10.1371/journal.pone.0016136 (DOI)000286519500041 ()2-s2.0-79251539925 (Scopus ID)
Funder
Swedish Research Council, 621-2004-4647
Note
QC 20110325Available from: 2011-03-25 Created: 2011-03-21 Last updated: 2011-05-16Bibliographically approved
2. Improved solubility of TEV protease by directed evolution
Open this publication in new window or tab >>Improved solubility of TEV protease by directed evolution
2006 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 121, no 3, 291-298 p.Article in journal (Refereed) Published
Abstract [en]

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV–GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Place, publisher, year, edition, pages
Amsterdam: Elsevier, 2006
Keyword
TEV protease; Directed evolution; GFP; Solubility; FACS
National Category
Biocatalysis and Enzyme Technology Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10962 (URN)10.1016/j.jbiotec.2005.08.006 (DOI)000235504800001 ()2-s2.0-30944459888 (Scopus ID)
Note
QC 20100729Available from: 2009-09-02 Created: 2009-08-31 Last updated: 2017-12-13Bibliographically approved
3. Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
Open this publication in new window or tab >>Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
2009 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 26, no 5, 251-259 p.Article in journal (Refereed) Published
Abstract [en]

Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.

Place, publisher, year, edition, pages
Amsterdam: Elsevier, 2009
Keyword
protein fragment complementation assay (PCA), beta-lactamase, survival selection, affibody affinity proteins, tumor necrosis factor-alpha
National Category
Biocatalysis and Enzyme Technology Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10964 (URN)10.1016/j.nbt.2009.06.980 (DOI)000272773400008 ()19576305 (PubMedID)2-s2.0-77950348500 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20100729

Available from: 2009-09-02 Created: 2009-08-31 Last updated: 2017-12-13Bibliographically approved
4. Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection
Open this publication in new window or tab >>Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection
2010 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, 111-120 p.Article in journal (Refereed) Published
Abstract [en]

The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.

Keyword
protein fragment complementation assay (PCA), β- lactamase, affinity maturation, affibody affinity proteins, tumour necrosis factor-α
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:kth:diva-10965 (URN)10.1042/BA20090274 (DOI)000276792800001 ()2-s2.0-77953131705 (Scopus ID)
Note
Uppdaterad från manuskript till artikel: 20100729 QC 20100729Available from: 2009-09-02 Created: 2009-08-31 Last updated: 2017-12-13Bibliographically approved

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