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Protein Engineering by Directed Evolution and Rational Design
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-5391-600X
2001 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH , 2001. , 87 p.
Keyword [en]
ion-exchange chromatography, subtilisin, Klenow DNA polymerase, Taq DNA polymerase, directed evolution, rational design, charge engineering, phosphonylating inhibitor, phage display, circular dichroism, protein A
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-3171ISBN: 91-7283-102-2 (print)OAI: oai:DiVA.org:kth-3171DiVA: diva2:8939
Public defence
2001-06-01, 00:00
Note
QC 20100609Available from: 2001-05-31 Created: 2001-05-31 Last updated: 2010-06-09Bibliographically approved
List of papers
1. Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
Open this publication in new window or tab >>Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
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1997 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-13273 (URN)10.1006/prep.1996.0674 (DOI)
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
2. Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors
Open this publication in new window or tab >>Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors
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2000 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 296, no 1, 87-102 p.Article in journal (Refereed) Published
Abstract [en]

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial Libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close Link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentils on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very Limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-L-Ala-L-Ala-L-Pro- Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-L-Lys-L-Ala- L-Pro-Phe(P)-diphenylester ter allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

Keyword
phage display, subtilisin, specificity, biotinylated inhibitors, enzyme selection, bacillus-lentus, intramolecular chaperone, escherichia-coli, mutational replacements, denatured subtilisin, assisted catalysis, directed evolution, crystal-structure, serine proteases, binding-proteins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-13274 (URN)10.1006/jmbi.1999.3437 (DOI)000085295900007 ()
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
3. Charge engineering of a protein domain to allow efficient ion-exchange recovery
Open this publication in new window or tab >>Charge engineering of a protein domain to allow efficient ion-exchange recovery
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2000 (English)In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, no 10, 703-709 p.Article in journal (Refereed) Published
Abstract [en]

We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

Keyword
circular dichroism, ion-exchange chromatography, molecular modelling, pI, protein A, bacterial receptor domain, escherichia-coli k-12, fusion protein, binding domain, nucleic-acids, force-field, purification, dna, resolution, sequence
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-13275 (URN)10.1093/protein/13.10.703 (DOI)000165995500006 ()
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
4. Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
Open this publication in new window or tab >>Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
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2002 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, no 1-2, 157-166 p.Article in journal (Refereed) Published
Abstract [en]

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8270 (URN)10.1016/S0021-9673(01)01413-3 (DOI)000173120900015 ()
Note
QC 20100609Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2017-12-14Bibliographically approved
5. Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
Open this publication in new window or tab >>Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
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2002 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, 93-102 p.Article in journal (Refereed) Published
Abstract [en]

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

Keyword
protein A, Z(basic), ion-exchange chromatography, expanded bed adsorption, DNA-POLYMERASE-I, ESCHERICHIA-COLI, PURIFICATION, RECOVERY, COXSACKIEVIRUS-B3, CHROMATOGRAPHY, EXPRESSION, EFFICIENT, CELLS
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-13276 (URN)10.1016/S0168-1656(02)00040-8 (DOI)000176318300010 ()
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved

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