Self-assembly of proteins and their nucleic acids
Research
Self-assembly of proteins and their nucleic acids
Graham Fletcher , Sean Mason , Jon Terrett and Mikhail Soloviev
Oxford GlycoSciences (UK) Ltd, Abingdon, Oxon OX14 3YS, United Kingdom
Journal of Nanobiotechnology 2003 1:1
The electronic version of this article is the complete one and can be found online at: jnanobiotechnology.com
Received 25 November 2002
Accepted 28 January 2003
Published 28 January 2003
© 2003 Fletcher et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
Keywords: self-assembly, protein, DNA, molecular engineering, molecular interface, cloning expression
Abstract
We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.
The 20th century has witnessed the birth of molecular biology and an explosion in cloning applications, the numbers of which exceeds hundreds of thousands. Traditional molecular cloning approaches are dependant on the ability of cells to both synthesise proteins from DNA and to replicate themselves and any exogenous DNA. This enables the linkage, within an individual cell, of the information-carrying DNA to the encoded protein or the cellular phenotype. Viruses and phages are also used in molecular biology and provide another means of "linking" protein (or protein function) to corresponding DNA but they are entirely dependent upon a host cell to replicate. Using cell- or phage-based cloning systems resolves a number of important problems. It allows the creation of a "one DNA vector per cell" system, which following a physical separation (by plating on a dish or through dilution) can be amplified (through self-replication) into a macroscopic colony which could then be catalogued, stored or grown further for preparative applications. However, the use of living cell-based systems has a number of disadvantages. Performing such experiments not only requires proper facilities, but they are also lengthy processes. Bacterial or phage cloning takes about a day to go from a single bacteria to a clone; yeast takes days to grow; and mammalian cells take weeks to form a clone. An adequate amplification of DNA can be achieved by other means. For the last decade PCR has been widely used instead of cloning for the production of large amounts of DNAs. However, no adequate system has so far been developed for linking the DNA, an information carrier, to its protein, a function carrier.
Direct linking of proteins to their DNAs or RNAs to bypass the limitation of cellular systems has been attempted before. One strategy has been to utilise components of the cellular protein synthesis machinery to transiently or permanently link mRNAs and proteins. Protein synthesis in living cells is a two-step process involving transcription, which is followed by translation. During transcription of DNA, an mRNA is made and processed by RNA polymerases and spliceosome complexes. Translation involves protein synthesis on ribosomes using mRNA as a template molecule. If transcription termination is blocked, the mRNA will remain in the complex with its DNA (and with the enzymes responsible for the RNA synthesis and splicing). Similarly, if translation termination is prevented the ribosome will remain associated with both the mRNA and the nascent protein chain. The discovery that the processes of transcription and translation could be performed outside the cell [1-3] has encouraged attempts to "link" such in vitro synthesised proteins to their nucleic acid. Taussig and He have employed the transcription-translation termination blockade to create transient {mRNA-ribosome-protein} complexes which physically crosslink the RNA with the associated proteins [4,5]. Such a "ribosome display" approach has a number of disadvantages, including the fact that the complexes obtained also include all elements of the protein synthesis machinery, i.e. ribosomes with all their associated RNAs and proteins. This not only depletes the translation reaction but also results in a very high background and large number of unrelated proteins linked to the mRNA. Xu et al [6] have produced intermediate {mRNA-DNA-adapter-ribosome-Protein} complexes where a puromycin-labelled DNA adapter, separately ligated to RNA molecules, covalently links to a nascent protein chain in a sequence-independent manner (an "mRNA display" approach, [6]). Such a modification results in covalent {mRNA-protein} complexes, which lack bulky ribosomes, but involve a high degree of non-specific crosslinking of the RNA to ribosomal proteins. Ligation of a puromycin-modified DNA to mRNA requires an additional step, which makes the whole procedure significantly longer especially if a few rounds of subsequent amplification and selection are required. A variation of RNA-protein complex production using puromycin was also reported by Roberts and Szostak, and by Liu et al [7,8] respectively. All the methods reported so far result in the production of covalently crosslinked protein-RNA hybrids and/or complexes containing bulky ribosomes or requiring multi-step processes and excessive RNA handling in order to make protein-DNA complexes. The use of mRNA in the techniques described above is disadvantageous because of the instability of RNA and its fast degradation compared to the more stable DNA molecules. Another disadvantage is the requirement for the two additional enzymatic steps, namely reverse transcription and cDNA amplification, before sequence information can be extracted.
Using a molecular scaffold of a streptavidin protein we have designed a protein vector – an interface synthesised in vitro, which contains a nucleic acid assembly module and a protein sequence of interest, thus providing a direct physical link between the expressed protein feature and its encoding DNA.
Figures
Figure 1
Design of the SAPV (streptavidin based protein vector)
Figure 2
Full length engineered nucleotide sequence (466 b.p.) coding for the SAPV protein vector
Figure 3
Nucleotide sequence of the tagged SAPV (1442 b.p.)
Figure 4
Detection of the tagged SAPV on Western blots
Figure 5
Assembly of the SAPV protein vector with biotinylated DNA
Figure 6
Structure of the streptavidin protein
Figure 7
Fragment of the core streptavidin amino acid sequence
Figure 8
BCMP84 immunoprecipitation on protein A-conjugated glass beads and on nitrocellulose membrane
Figure 9
Co-immunoprecipitation of the assembled SAPV-84
Figure 10
Immunoprecipitation of the self-assembled SAPV vectors
Tables
Table 1
Engineered SAPV displaying Albumin and BCMP84 peptides.
Design of a protein vector based on the core protein sequence of streptavidin (SA)
Streptavidin (from Streptomyces avidinii) is a naturally occurring protein, which is able to bind biotin (Figure 1A) with high affinity. The nucleotide sequence of the streptavidin gene was reported in 1986 by Argarana et al [9]. We have used the Streptomyces avidinii gene for streptavidin (X03591, Figure 1C) as a scaffold for designing a streptavidin based protein vector (SAPV, Figure 1B). Full length nucleotide sequence coding for the SAPV (Figure 2) was produced using overlapping synthetic oligonucleotides (obtained from Sigma-Genosys) and several rounds of PCR (for oligonucleotide primers and details of the synthesis see Materials and Methods). For efficient transcription by bacterial T7 polymerase, two T7 RNA polymerase binding sites and a T7 terminator sequence were inserted into the engineered SAPV DNA. It also contained a ribosome-binding site (RBS) – a signal necessary for efficient translation, see Figure 2. SAPV DNAs for use in the in vitro Transcription/Translation (T&T) were routinely obtained by PCR (see Methods). To confirm efficient expression of the SAPV at the protein level, the SAPV was designed with a protein tag (autofluorescent protein AFP). The engineered nucleotide sequence of the tagged SAPV is shown in Figure 3. Tagged SAPV DNA was generated in the same way as the untagged SAPV DNA. Tagged SAPV was detected on Western blots with anti-GFP Rabbit polyclonal antibody, see Figure 4. The strong staining confirmed efficient synthesis of the SAPV-AFP. Based on the results of this experiment, the optimal experimental conditions for all subsequent T&T reactions included the use of 2 ug DNA per 20 ul of the in vitro T&T reaction, the synthesis temperature was maintained at 21°C.
To control whether SAPV protein vector is able to bind biotinylated DNA, a completed T&T reaction was incubated with either biotinylated or non-biotinylated DNA. The longer DNAs were chosen for assembly reactions to avoid non-specific background due to the SAPV DNA used in the in vitro T&T reaction. Protein-DNA complexes were separated from free DNAs by filtration through a protein-binding filter and the retained DNAs were detected by PCR. The amplified products were separated on agarose gels. Equal amounts of each PCR reaction were loaded onto each lane (Figure 5). The absence of a signal in the 4th wash (in both the biotinylated and non-biotinylated DNA assemblies) confirms the absence of a non-specific background. The eluates from the biotinylated DNA experiments (Figure 5A,5C) contained large amounts of amplified DNA, whilst the eluates from the non-biotinylated DNA assemblies (Figure 5B,5D) did not. This clearly demonstrates that the designed SA-based tagged protein vector is able to bind biotinylated DNAs.
Assembly and affinity precipitation of SAPVs displaying a BCMP84 peptide
The core protein sequence of streptavidin and the streptavidin-based SAPV contains a 9 amino acid long loop (GTTEANAWK, Figures 6 and 7), which we predicted to be most suitable for modifications, such as SAPV extension, modification, or for expressing other protein fragments, peptides and tags. This choice is based on the molecular architecture of streptavidin (Figure 6B). To illustrate the "display" capabilities of the SAPV, we have engineered SAPV-Alb5 and SAPV-84 which display peptide fragments of Albumin and BCMP84 proteins, respectively (Table 1). The choice of the peptides was determined by the antibodies available (polyclonal anti-albumin antibody, which recognise the Albumin peptide, and polyclonal anti-BCMP84 anti-peptide antibody). DNAs encoding the modified SAPV (SAPV-Alb5 or SAPV-84) were obtained by PCR. A co-immunoprecipitation system was designed to quickly separate different SAPVs. The protocol was tested using a recombinant BCMP84 protein. We separately tested glass bead-based and nitrocellulose-based systems. Comparable amounts of BCMP84 protein were present in the eluates from both the beads and the nitrocellulose, indicating that the protein was selectively retained (Figure 8).
Assembled SAPV-84 protein-DNA complexes were immunoprecipitated using either anti-BCMP84 or anti-albumin antibodies bound to nitrocellulose. Following a number of washes, the SAPVs were eluted and the eluates assayed by PCR amplification of the SAPV-84 DNA. The results of 5 independent measurements are presented in Figure 9. The results indicate that immunoprecipitation of SAPV-84 on the anti-BCMP84 nitrocellulose is significantly higher than on the control anti-Albumin nitrocellulose. The approximately 2.5x fold difference cannot be taken as a fully quantitative measurement as this assay employed an end point PCR detection, which may have gone out of the logarithmic amplification phase. However, the clear predominance of the assembled SAPV-84 in the eluate from the anti-BCMP84 nitrocellulose confirms that the BCMP84 peptide was adequately displayed on the SAPV-84 protein vector, which was assembled with the biotinylated SAPV-84 DNA and precipitated by anti-BCMP84 antibody.
Self-assembly of protein vectors with their DNAs and affinity separation
Co-transcriptional and co-translational self-assembly of SAPV protein vectors with their encoding DNAs is demonstrated using SAPV-84, SAPV-Alb5 and "empty" SAPV-only (unmodified) protein vectors. The in vitro synthesised and assembled SAPVs were incubated with either anti-BCMP84 or anti-Albumin antibodies, which were immobilised on beads. Following incubation and washings, the co-immunoprecipitated SAPVs were eluted and assayed by PCR. Equal amounts of each PCR reaction were analysed by electrophoresis (see Figure 10). The figure clearly demonstrates that only correct self-assembled SAPVs are precipitated, i.e. SAPV-84 DNA is co-precipitated on anti-BCMP84 beads and SAPV-Alb5 DNA is co-precipitated on anti-Albumin beads.
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