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Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
2002 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 93, no 1, 1-14 p.Article in journal (Refereed) Published
Abstract [en]

Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated, Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M 13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.

Place, publisher, year, edition, pages
2002. Vol. 93, no 1, 1-14 p.
Keyword [en]
aqueous two-phase system; peptide library; partitioning; phage display; selection; recombinant proteins; fusion tag; PEG/sodium phosphate system
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-6923ISI: 000173186700001OAI: oai:DiVA.org:kth-6923DiVA: diva2:11773
Note
QC 20100623Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
Open this publication in new window or tab >>Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Bioteknologi, 2007
Keyword
Aqueous two-phase system, combinatorial library, expression vector, flow cytometry, fusion tag, partitioning, peptide library, phage display, recombinant proteins, ribosomal binding site, translation initiation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4318 (URN)978-91-7178-607-4 (ISBN)
Public defence
2007-04-20, Sal F3, KTH, Lindstedtsvägen 26, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100623Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2012-03-19Bibliographically approved

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Nygren, Per-Åke

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