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Strategies for facilitated protein recovery after recombinant production in Escherichia coli
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0140-419X
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.

A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger.

Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography

Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer.

The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , p. 91
Keywords [en]
ion-exchange chromatography, protein A, Z, Zbasic, Zacid, fusion protein, proteolytic cleavage, immobilised protease, flow cytometry, inclusion bodies, solid-phase refolding
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-471ISBN: 91-7178-176-5 (print)OAI: oai:DiVA.org:kth-471DiVA, id: diva2:13554
Public defence
2005-11-18, Sal FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:15
Opponent
Supervisors
Note
QC 20101020Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2018-01-13Bibliographically approved
List of papers
1. 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, p. 157-166Article 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
2. 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, p. 93-102Article 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.

Keywords
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
3. Negatively charged purification tags for selective anion-exchange recovery
Open this publication in new window or tab >>Negatively charged purification tags for selective anion-exchange recovery
2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 11, p. 779-786Article in journal (Refereed) Published
Abstract [en]

 A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

Keywords
ion-exchange chromatography, protein A, protein design, Z domain
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8272 (URN)10.1093/protein/gzh092 (DOI)000227133500003 ()2-s2.0-14644444571 (Scopus ID)
Note
QC 20100923 QC 20110915Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2017-12-14Bibliographically approved
4. A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein
Open this publication in new window or tab >>A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein
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2005 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, no 2, p. 133-146Article in journal (Refereed) Published
Abstract [en]

A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

Keywords
protein production levels; inclusion bodies; green fluorescent protein (GFP); flow cytometry; Z(basic); His(6)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-8273 (URN)10.1016/j.jbiotec.2005.03.024 (DOI)000232042900003 ()2-s2.0-24044436165 (Scopus ID)
Note

QC 20100824

Available from: 2005-11-02 Created: 2005-11-02 Last updated: 2017-12-14Bibliographically approved
5. Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag
Open this publication in new window or tab >>Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag
2006 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 1, p. 187-196Article in journal (Refereed) Published
Abstract [en]

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

Keywords
Zbasic, Ion exchange chromatography, Purification of inclusion body proteins, Solid-phase refolding
Identifiers
urn:nbn:se:kth:diva-12815 (URN)10.1002/biot.200500023 (DOI)16892247 (PubMedID)2-s2.0-33750601043 (Scopus ID)
Note
QC20100622Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2010-10-20Bibliographically approved
6. Enzymatic cleavage of fusion proteins using immobilised protease 3C
Open this publication in new window or tab >>Enzymatic cleavage of fusion proteins using immobilised protease 3C
2006 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 47, no 2, p. 422-426Article in journal (Refereed) Published
Abstract [en]

A strategy for efficient cleavage of fusion proteins using an immobilised protease has been developed. Protease 3C from coxsackie virus was recombinantly produced in Escherichia coli and covalently immobilised onto a solid support. Thereafter, Z(basic) tagged fusion proteins, with a specific cleavage sequence between the domains, were flown through the proteolytic column and circulated until complete cleavage. Subsequently, the processed protein solution was applied on a cation exchanger. Thereby, removal of the released, positively charged fusion tag, Z(basic), was done by adsorption to the matrix while the target proteins were recovered in the flow through. Interestingly, the columns were shown to be reusable without any measurable decrease in activity. Moreover, after storage in 4 degrees C for two months the activity was almost unaffected.

Keywords
immobilised protease, Z(basic), protease 3C, cleavage, purification, efficient
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-15745 (URN)10.1016/j.pep.2006.01.003 (DOI)000238277000011 ()2-s2.0-33646541425 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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