Oktober 2012
Picha pastoris expressing recombinant spider silk, is it possible?
Cheuk Hin Lau
UPTEC X 12 019 Date of issue 2012-06 Author
Cheuk Hin Lau
Title (English)
Picha Pastoris expressing recombinant spider silk, is it possible?
Title (Swedish)
Abstract
Native spider silk is a versatile biomaterial that is stronger than steel, tougher than Kevlar and yet very flexible. This combined with recent research showing biocompatibility with cells gives the spider silk a wider range of applications such as for biomedical applications. A total of 5 different genes (A, B, C, D, E) were cloned into P. pastoris, with each gene containing 4RepCT coupled to different tags. The genes were ligated to expression vectors pGAPZαC, pGAPZαA and pPICZαA and were successfully transformed to P. pastoris. So far gene B has been successfully expressed and secreted by P. pastoris using the expression vector
pPICZαA.
Keywords
Pichia pastoris, Spider silk, Cloning, Protein expression, Purification, Protease assay Supervisors
My Hedhammar & Mats Sandgren
Swedish University of Agriculture Sciences (SLU)
Scientific reviewer
Margareta Krabbe
Uppsala University
Project name Sponsors
Language
English
Security
Secret until 2014-12
ISSN 1401-2138 Classification Supplementary bibliographical information Pages
48
Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 471 4687
Molecular Biotechnology Program
Uppsala University School of Engineering
Picha Pastoris expressing recombinant spider silk, is it possible?
CHEUK HIN LAU
POPULÄRVETENSKAPLIG SAMMANFATTNING
Naturlig spindeltråd är ett mångsidigt biomaterial som är starkare än stål, tåligare än kevlar och ändå mycket flexibel. Detta i kombination med den senaste forskningen som påvisar dess biokompatibilitet ger spindeltråd ett bredare spektrum av möjliga
applikationer till exempel inom biomedicin.
Genom rekombinant uttryck av en liten del av spindeltrådsprotein (4RepCT) i E. coli, har proteiner som själv sammansätts till mikroskopiska fibrer med liknande egenskaper som det nativa spindelsilke fibrer åstadkommits. Emellertid har 4RepCT en tendens att bilda proteinaggregat, vilket försvårar hantering av proteinet.
Målet för detta examensprojekt var att klona, uttrycka och utsöndra 4RepCT i Pichia pastoris. Fem olika gener (A, B, C, D, E) klonades in P. Pastoris. Generna ligerades till expressionsvektorerna pGAPZαC, pGAPZαA och pPICZαA som därefter framgångsrikt klonats till P. pastoris. Flera försök med att uttrycka pGAPZαC och pGAPZαA vektorn i P.
pastoris prövades, dock så kunde inte målproteinet upptäckas. Ändring av expressionsbetingelser genom att ändra temperatur, pH och buffert löste inte problemet. Detta visade sig senare bero på proteolytisk nedbrytning orsakad av P.
pastoris specifika proteaser som finns närvarande under odlingen. Genom att tillsätta proteashämmare till odlingsmediet så kunde vi på så sätt uttrycka och utsöndra gen B produkten framgångsrikt med expressionsvektorn pPICZαA.
Examensarbete 30 hp
Civilingenjörsprogrammet Molekylär bioteknik Uppsala universitet, Oktober 2012
Table of contents
Introduction ...
Spider silk ... 9
Spider silk secretion pathway of dragline silks ... 9
Spider silk applications ... 10
Recombinant spidroin production in different host system ... 11
Pichia Pastoris ... 13
Description and aim of the project ... 15
Material and methods ... Gene and expression vector ... 16
Infusion cloning procedure ... 16
T4 ligase cloning procedure ... 17
pPICZαA cloning procedure ... 17
Sequence analysis ... 18
Pichia pastoris transformation procedure ... 18
Expression procedure ... 18
Protein purification (supernatant and cell lysate) ... 19
Supernatant ... 19
Cell lysate ... 20
Protease assay ... 20
Cloning and expression results ... Infusion cloning to pGAPZαA ... 21
T4 ligase to pGAPZαC ... 22
pPICZαA cloning ... 25
Sequence analysis ... 26
Transformation to Pichia pastoris ... 27
Expression analysis of expression vector pGAP ... 28
Low temperature expression of vector pGAP ... 29
Protein expression at high pH of vector pGAP ... 30
High pH and low temperature expression of vector pGAP ... 31
Cell analysis ... 32
pPic expression ... 33
Protease assay results ... Papain protease assay ... 35
Growth media protease assay ... 36
pH adjusted growth media protease assay ... 37
Pepstatin A and EDTA protease assay ... 38
Additional results ... Solution to chelation of the IMAC column ... 39
Discussion ... Cloning and transformation difficulties ... 40
Expression difficulties using pGAP vector ... 41
Interpretation of the results obtained from protease assays ... 41
Successful expression using pPIC vector ... 42
Comparison between pGAP and pPIC ... 42
Alternative solutions for protease activity ... 43
Advantages of P. pastoris ... 43
Other obstacles ... 43
Future work ... 43
Acknowledgements ... 44
References ... 45
Key terms and abbreviations
4RepCT C-‐terminal domain and four repetition of the repetitive region of the spidroin.
AOX1 One of two genes that code for the enzyme alcohol oxidase in Pichia pastoris.
AOX2 Second gene that codes for the enzyme alcohol oxidase in Pichia pastoris.
ARG4 A S. cerevisiae gene, host with this gene has the function of growing in media lacking arginine.
Dragline silk One type of spider silk.
Endo H Deglycosylation enzyme
GAP Glyceraldehyde-‐3-‐phosphate dehydrogenase gene
HIS4 Histidinol dehydrogenase gene
MaSp1 Major ampullate spidroin 1
MaSp2 Major ampullate spidroin 2
Mut-‐ A phenotype of Pichia pastoris that has the AOX1 gene largely
deleted and replaced with the S. cervisiae ARG4 gene.
Mut+ A phenotype of Pichia pastoris that has both of the genes AOX1
and AOX2 present in their genome.
pGAPZαA Expression vector supplied by Invitrogen.
pGAPZαC Expression vector supplied by Invitrogen.
pPICZαA Expression vector supplied by Invitrogen.
PTM Post translation modification
Spidroin Protein that assembles into spider silk and are comprised of 3000-‐4000 amino acids, that are composed of 3 parts: an N-‐
terminal domain, a large repetitive region and a C-‐terminal domain.
Introduction
SPIDER SILK
Spider silk is by far the toughest natural fibers known to mankind [2], and this naturally produced biomaterial has properties that exceeds todays synthetic materials. For
example silk is as much as 5 times stronger by weight than steel and have physical properties comparable to those of the synthetic fiber Kevlar [3]. Using spider silk to our advantage has been done for many centuries, it has been ascribed to stop bleeding and promote wound healing. Other appealing properties of spider silk include
biodegradability [4], biocompatibility [5], stickiness and flexibility [6]. Thanks to
different molecular bio-‐techniques the mysterious biochemical properties of spider silk has been partly solved. This incredible silk is comprised of at least two similar proteins, the major ampullate spidroin 1 (MaSp1) and ampullate spidroin 2 (MaSp2). The ratio between MaSp1 and MaSp2 in spider silk has been estimated to be 3:2 [6].
The spidroin proteins are macromolecules comprised of 4000-‐3000 amino acids [7] that can be divided into 3 parts, an N-‐terminal domain of approximately 130 amino acids, a C-‐terminal domain of approximately 110 amino acids and a large repetitive region, which is flanked by the N-‐terminal and C-‐terminal, of approximately 3500 amino acids that is composed of poly-‐alanine blocks and glycine rich segments [8] (fig1). The N-‐
terminal is highly conserved trough different spider spices and the domain is not a necessity when forming synthetic spider silk, but is highly soluble which may be an important factor for spidroin post expression processing [9]. The C-‐terminal domain governs the spider silk assembly while preventing unwanted aggregation [10]. The repetitive region is what gives the spider silk its strength and flexibility when assembled. The secondary
structure of the repetitive region converts mainly into β-‐sheet during spider silk formation. While in soluble form the spidroin
contains mainly random and α-‐
helical structures [9]. Depending on different species of spider, the amount of glycine and alanine will
differ in the repetitive region and hence the strength and mechanical properties of the spider silk fibers will also differ [11].
SPIDER SILK SECRETION PATHWAY OF DRAGLINE SILK
The mechanism behind production and secretion of spider silk protein in spiders is quite well understood. The pathway of secretion can be divided into two zones, A-‐zone and B-‐zone [3].
The A-‐zone contains the tail and first
Figure 1: Schematic picture of the composition of spidroin. The spidroin protein are divided into 3 parts, a repetitive region, an N-terminal and a C-terminal.
Figure 2: Schematic representation of the major ampullate gland in orb spiders.
part of the sac. Here a single-‐layered epithelium produces the spidroins that are
exocytosed into the lumen (tail) [12]. Spidroins that are secreted into the lumen can be stored at a very high concentration (30-‐50% W:V) in the sac until they are converted into solid fibers when needed [13]. The spinning duct has an s-‐shaped form and gets narrower while approaching the end of the duct. Here the spidroins assembles into fibers with the help of pH adjustment, changes in ion composition and shear force along the duct [3] (Figure 2). Each gland produces a specific type of spider silk [1]. Orb spiders have seven different types of glands and the ampullate gland produces only dragline silk [1].
SPIDER SILK APPLICATIONS
Spider silk is biodegradable and has trough history been ascribed for use in medical applications. Thanks to these amazing features many researchers lately have proposed spider silk to be the ideal biomaterial [14]. The understanding of spider silk proteins has just started in the recent decades, which makes the studies for using spider silk as
biomaterial relative few. But many ideas have been proposed for the use of spider silk as biomaterial and spider silk proteins have shown to have a large potential in many
different biomedical applications [14].
The main studies of spider silk today are to use of this for biomedical applications. But different applications put different and sometimes more complex demand on the produced spider silk material. The term biomaterial is often mentioned along with biocompatibility. The definition of biocompatibility consists basically of two
components: bio functionality and biosafety [14]. Definition of biosafety is for example appropriate local response or lack of systemic response and absence of cytotoxic and mutagenesis/carcinogenesis [14]. Biofunctionality is for example the ability of the biomaterial product to perform its intended task/tasks [15]. One have to keep in mind that the definition might vary a lot, because the biomaterial might have a different effect on one tissue compared to another, and depending on where the biomaterial is going to be applied, the degree for inflammatory reaction will vary [14]. Furthermore the
requirement for the spider silk are changed depending if its intended to be used in vitro or in vivo [14].
If the usage of spider silk as a biomaterial will have any chances to come true in the future, the question that has to be answered is if recombinant spider silk is
biocompatible [14]. There have been several studies to assess the biocompatibility with living tissue. One such study is to use recombinant spider silk proteins to form a porous membrane and investigate if that membrane could act as a wound dressing on deep burns in rats [16]. The results of the study was that the recombinant spider silk had a good biocompatibility and had the ability to induce tissue regeneration, according to the article as efficient for wound healing as clinically used collagen sponges [16]. This shows that spider silk proteins have good prospects as a new type of biomaterial for tissue-‐
engineering of artificial skin [16]. Another study that was done on recombinant spider silk protein was to see how well the body of a rat did accept the spider silk fibers when implanted subcutaneously [17]. The study did show that the recombinant spider silk protein was well accepted, and newly formed capillaries and fibroblast-‐like cells in the center of the silk was observed already after one week of implantation, which indicates that the spider silk supports the formation of vascularized tissue [17]. According to the paper presenting these results the results looks promising, but further in vivo studies
have to be performed to fully evaluate the ability of spider silk as a biomaterial for tissue engineering in humans. Similar results have also been observed in another study, were spider silk protein in form of porous bar shaped scaffolds were implanted
subcutaneously in mice [18]. The result in this study did show that the scaffold had good biocompatible properties in vitro and in vivo. It was also observed eight weeks after implantation that the scaffolds promoted ingrowth of fibrous, nerve cells and adipose tissue elements [18]. The conclusion from the study was that the spider silk scaffold could be applied in biomedical tissue engineering [18].
Another application that has been investigated using recombinant spider silk is to make film for cell attachment and proliferation in vitro for cell cultures and biomedical sensors [19]. The different types of spider silk film, foam, fiber or mesh scaffold offer both 2D and 3D cell culture environments which have shown to support attachment and growth of human primary fibroblasts [19]. The article also stated that the spider silk matrix proved to be robust and different types of spider silk offers a similar support of cell growth. Other benefits of using recombinant spider silk as a cell adhesion matrix would be to modify this with a certain adhesion binding molecules added in the spider silk protein sequence during expression [14]. Many cells interacts with the environment via adhesion molecules such as: integrins, cadherins and selectins [14]. If these adhering molecules are added to the spider silk protein, the 3D silk matrix will be able to mimic a more natural environment for the cells. These functionalized spider silk matrix could have the possibility to provide the cells with signals for differentiation, growth and migration. One study shows that by introducing an integrin binding motif RGD into the spider silk, it supported and further enhanced the growth of bone tissue [20].
Another interesting usage of recombinant spider silk protein was shown in an study were the researches was testing it as a drug carrier [21]. The aim in this study was to make a drug carrier that has the function of a controlled delivery of positively charged and sufficiently hydrophobic drug molecules. The molecules were loaded onto the spider silk by hydrophobic and electrostatic interactions and were slowly releases from the surface, which lead to a constant drug release rate. The authors of the paper did conclude that the spider silk particles have diverse applications where this type of release and mechanically tough and slowly biodegradable carriers is desired.
Applications for spider silk protein are not confined for biomedical application. Other areas could benefit of using spider silk protein, such as cosmetic products, shampoos, soap creams and nail varnish [22]. The spider silk protein will lead to enhancement of the softness, brightness and toughness of the product. For technical applications the spider silk has a potential to be used in micro-‐mechanical and electronic set-‐ups [22].
Inorganic particles such as metals could be incorporated into spider silk nano-‐fibrils, and these crossover materials could be used for nanowires or surface coatings. Lastly the properties that spider silk possess and the similarity with the silk from Bombax-‐
mori, points towards that spider silk can be applied and used in technical textiles, for example parachute or even bulletproof vest. Textiles that have a high demands on toughness and strength [22].
RECOMBINANT SPIDROIN PRODUCTION IN DIFFERENT HOST SYSTEM
Because of the amazing biochemical properties that spider silk posses, researchers have tried many ways to produce spidroins recombinantly in different host systems, both in
prokaryotic and eukaryotic hosts [13]. There are several big disadvantages of using spiders for spider silk production, even though they have the mechanism ready to be used. Spiders in nature are cannibals, which means they cannot live in the same cage [23], the spider also produce low yield of spider silk protein and could not be collected easily [24]. That is why scientist has chosen to focus on alternative ways to produce spidroins [13]. Different host systems have their own pros and cons, such as: difference in expression levels, ease of use, contamination levels during production and most important for industrial purpose; cost compared to protein yield [13].
Many aspects have to be taken into account when choosing a suitable protein expression host. Spidroins are eukaryotic proteins, and eukaryotic proteins often require post-‐
translation modifications in order to get a correct fold and biological activity [25]. Most of mechanisms for modifications (glycosylation, phosphorylation etc.) are only present in eukaryotic cells, and different eukaryotic cells have different modifications pattern which could affect the spider silk properties. Other complications are the nature of the spidroins itself. As mentioned above, the repetitive sequence of spidroins is composed of poly-‐alanine blocks and glycine rich segments, which could lead to tRNA-‐pool depletion in the host [26]. On DNA level the repetitive region is very rich in guanine/cytosine and this has been shown to be problematic when expressing a protein recombinantly in another host than the original one [27]. Genetic instability, mRNA forming unwanted secondary structures, truncations and rearrangements of the gene during duplication and translation pauses [27] is some of the problems that has occurred during
recombinant protein expression. Further on, low solubility of the spidroins cause them to easily form aggregates [13]. The proteases expressed by the expression host might also degrade the spidroins [28]. Approaches to overcome these obstacles have been tried, such as codon optimization [27] and culturing in enriched media.
Although eukaryotic hosts might be the most suitable for recombinant production of spidroins, there are several reports of successful recombinant production of fractional parts of spidroins in prokaryotes [26, 27]. The most broadly used prokaryotic host is the bacterium Escherichia coli, although it has drawbacks, such as: low yield of protein, protein accumulation in the cell, and instable protein fragments [13]. E. coli offers a well-‐
controlled, cost-‐efficient system for large-‐scale production and several cases of successful E. coli expression processes have been published [27]. On top of that, the expression system of E. coli is well studied and easy to handle [29].
Another host that has been used for recombinant production of spidroins is the yeast Pichia pastoris (P. pastoris). The advantage of using yeast instead of E. coli for
recombinant spidroin expression is to minimize truncations due to translation stop [30].
P. pastoris is a eukaryotic host, which means larger and more complex fragments of spidroins will be possible to produce [30]. A fractional part of the repetitive part of spidroins has been reported to be successfully expressed intracellulary in P. pastoris.
The part that was successfully produced was a short segment of the repetitive region of the spidroin [30-‐32], and it was noted that the gene was duplicated in the genome, which resulted in varied sizes of target protein [30]. However according to the papers, the results from the spidroin production in P. pastoris was better compare to E. coli in many aspects, such as protein yield. P. pastoris produced almost 1g/l of target protein which is at least two fold higher then the expression levels observed when using E. coli [30], and even higher protein expression levels should be possible to reach when using P. pastoris expression host. Additional advantages with P. pastoris compared to E. coli is
that P. pastoris is able to produce more complex and larger proteins, up to 3000 amino acids in P. pastoris compared to 1000 amino acids in E. coli [30]. By fusing an
appropriate secretion signal when expressing the spidroin in P. pastoris the target protein can be secreted to the growth media [29], and the spidroin that has been successfully secreted is the same spidroin that has been produced intracellular in P.
pastoris. However it was noted in the study that a lot of the target protein stayed inside the cell [29]. Secretion will be an essential characteristic for any low-‐cost silk production process, not just because of the huge advantages on downstream process such as
purification and recovery of the product, because the amount of expressed protein that is needed to give a cost-‐effective production yield would overwhelm the available intracellular volume of the expression host [29].
Different and more complex hosts have been tested for the expression of spidroins.
Various mammalian cells have been attempted to express high-‐molecular-‐weight spidroins from bovine mammary epithelial alveolar cells to baby hamster kidney cells grown in a hollow fiber reactor [33]. Secretion of spidroins into milk of goats and mice in their glands has been successfully accomplished, but the yield was low compare to the high production cost and the time that was consumed to perform the expression [34, 35]. Expression in different plants such as potato, tobacco and Arabidopsis, has been tried as an attempt to have a low-‐cost efficient production suitable for scale-‐up.
However, the attempts of large-‐scale productions have only resulted in low yield of spidroins [22, 24]. Insect cells have been used for production of spidroins, but primarily for the study of assembly properties of spidroin pieces in the cytoplasm since insects cells are less suitable for large-‐scale production [36]. Last but not least expression in larvae of the silkworm Bombyx mori, have been tried and the target protein got
expressed but the amount of product was limited by the solubility of the spidroins [14].
PICHIA PASTORIS
The eukaryotic cell Pichia Pastoris (P. pastoris) is a methylotrophic yeast that can utilize methanol as a sole source of carbon as energy. It was discovered less then 50 years ago by Koichi Ogata [37]. Because of the properties that P. pastoris posses, such as capable of performing posttranslational modifications performed by higher eukaryotic cells and also cheaper, faster and easier to use then other eukaryotic systems, such as baculovirus or mammalian tissue culture [38], it has rapidly been accepted as a system for
expression of heterologous proteins [39]. With the right secretion signal peptide coupled to the expressed, target protein can be expressed extracellulary [40].
P. pastoris is one of a dozen known yeast species that are capable of metabolizing methanol [41]. If methanol is fed to P. pastoris as carbon source, the methanol will be processed by an enzyme called alcohol oxidase (AOX) that is coded by two genes AOX1 and AOX2 in P. pastoris [42]. AOX1 is responsible for the majority of AOX activity in the cell, and the AOX1 promoter is strictly regulated and induced by the levels of methanol in the surrounding culture liquid [42]. This regulating feature is one of the benefits of using P. pastoris as an expression host. There are several different commercial P.
pastoris expression strains available today that can be used for expression of
heterologous proteins [40]. These P. pastoris strains are different in terms of their ability to utilize methanol, selection markers to allow selection of expression vectors and also lack of native protease expression [38]. Most of the strains have a mutation defect in histidinol dehydrogenase gene (HIS4), that allows for selection of expression vectors
containing the HIS4 gene upon transformation, which will restore the function of growing in media lacking histidine [38]. Other variants of host strains are deletions of one or both AOX genes that will affect their ability to utilize methanol [38]. These P.
pastoris AOX deletion strains are sometimes shown to be better at producing foreign proteins [43]. The Result of deletion of AOX genes will lead to different phenotypes of P.
pastoris, one phenotype Mut+ has both of the genes AOX1 and AOX2 present in their genome and grows in methanol at wild-‐type rate [40]. Another phenotype, Mut-‐, has the AOX1 gene largely deleted and replaced with the S. cervisiae ARG4 gene, this will restore the function of growing in media lacking arginine and will act as a selection marker [38], and the strain will have to rely on a weaker AOX2 gene for AOX activity which
tremendously limits its growth rate [40]. Lastly there are strains that have both of the AOX gene removed and is not able to grow on methanol [40].
Endogenous proteases in P. pastoris could pose problems for expressed foreign proteins, since the protein of interest could be degraded by these proteases. Major vacuolar proteases appear to be a significant factor in degradation [40]. Because of the high cell density during P. pastoris cultivation a small percentage of the cells will get lysate and thereby releasing the protease to the growth media, which especially pose a problem for secreted protein [44]. But there are strains that have been modified to be defective in proteases, which have proven to help reduce degradation in several cases and
significantly improve overall yields [39].
Plasmid vectors are used for cloning target genes into P. pastoris, and these vectors have several features common. For methanol induced expression the cassette is composed of a DNA sequence containing the AOX1 promoter, followed by several digestion restriction sites, digestion restriction sites are DNA sequence are targeted by a given restriction enzyme that cleaves the DNA fragment [45]. Some digestion restriction site are designed to be unique to that specific expression vector and are used for insertion of the target gene and sequence coding [40]. This is followed by the transcriptional termination sequence from the P. pastoris AOX1 gene that directs efficient 3’ processing and
polyadenylation of the mRNAs [40]. Other more specific features may include sequences required for plasmid replications and maintenance in bacteria or AOX1 3’ flanking sequence that can help the foreign gene cassette in the expression vector to integrate at the AOX1 locus in P. pastoris genome by gene replacement [40]. Other differences also include selectable markers such as HIS4 gene and drug resistance markers against Zeocin, kanamycin et cetera [40]. There are also vectors that are design to be able to construct multiple expression cassette copies into one single vector. These vector are attractive if the aim is to have a high gene expression during induction [40]. Methanol induced expression is not the only available option, the GAP promoter is derived from the P. pastoris glyceraldehyde-‐3-‐phosphate dehydrogenase gene and is sometimes used instead of the AOX1 promoter [39]. The GAP promoter gets induced by glucose and is a suitable alternative if for some reason methanol cannot be used during expression [39].
One of the biggest advantages using P. pastoris as an expression host is that by infusing a secretion signal in front of the target protein in the protein will be secreted to the
growth media during expression [39]. Because the amount of endogenous secreted proteins by P. pastoris is very low, the majority of the protein in the growth media will be the protein of interest [40]. Several different secretion signal sequences have been used successfully, with S. cerevisiae α-‐mating factor with the most success [40].
When an expression vector gets introduced to P. pastoris, there are two different ways
that this can be integrated to the genome of P. pastoris [40]. Depending of the design of the vector either single crossover integration or gene replacement will occur [38]. DNA fragment of AOX1 or GAP promoter in the expression vector match the fragment that P.
pastoris carries and contains a specific restriction site. The restriction site in the
expression vector can be used to linearize the vector and aid the vector to integrate into P. pastoris genome during transformation [38]. Sometimes multiple gene insertions can be detected but at very low frequency, this could happen in all the gene insertion events [46].
Post translation modifications (PTM) are present in all higher eukaryotic hosts and that includes P. pastoris [40]. These PTM includes processing of signal sequences such as:
secretion signals, folding of protein, disulfide bridge formation and O and N-‐liked
glycosylation [40]. Many of these PTM is usually associated with higher eukaryotes [40], and this gives P. pastoris a larger advantage compare to other lower eukaryotic hosts.
Even tough glycosylation can be performed in P. pastoris, it have been shown to be problematic to replicate glycosylation that occurs in mammalian cells [47]. Unlike
mammalian cells where O-‐linked oligosaccharides are composed of variety of sugars, the O-‐oligosaccharides that are attached to proteins expressed by P. pastoris consist only of mannose residues [47]. For N-‐glycosylation there has also been observed difference between higher eukaryotic hosts and P. pastoris [39]. Additionally, P. pastoris can O-‐
glycosylate proteins that normally are not glycosylated in its native host, and foreign proteins that are secreted by P. pastoris can occasionally became hyper glycosylated [39]. This hyper glycosylation could pose a problem during SDS-‐PAGE characterizations where the protein appears to be of a different size.
DESCRIPTION AND AIM OF THE PROJECT:
A total of 5 different spider silk genes will be cloned into Pichia pastoris, each gene containing the 4RepCT gen, which are the C-‐terminal domain and four repetition of the repetitive region of the spidroin. The 4RepCT gen construct will be coupled with different tags, the tags will mainly serve as a purification tag, but will hopefully also increase the solubility of minispidroin and thus solve the problem of protein
aggregation. The target genes will be ligated to the P. pastoris expression vectors pGAPZαC, pGAPZαA and pPICZαA, which will then be transformed to P. pastoris for expression and secretion of the 4RepCT protein. Different modifications will be tried during expression of the target protein 4RepCT, such as alternating the cultivation temperature, growth media, expression vector etc.
Materials and methods
GENE AND EXPRESSION VECTOR
For this project 5 different 4RepCT genes (denoted A, B, C, D, E) were used for ligation into different P. pastoris expression vectors. Primers were design to generate PCR fragments of the genes A, B, C, D and E, all containing the part 4RepCT but coupled with different tags, from template vectors containing such sequences. Three different P.
pastoris expression vectors supplied from Invitrogen were used. The expression vector pGAPZαA was used for genes A, B, C, pGAPZαC for genes B, D, E and pPICZαA for gene B and E.
Infusion cloning was used for gene ligation of A, B, C into the pGAPZαA expression vector. Following the protocol of infusion cloning (Clontech), both the gene and the vector were amplified using PCR amplification techniques. Both forward and revers primer (DNA technology) for the vector contains 21 nucleotides that are complementary to part of the pGAPZαA vector. A forward gene primer (DNA technology) with a 15 nucleotide long sequence that is complementary to the revers vector primer was
introduced to the 3’end, whereas 15 nucleotide sequence complementary to the forward vector primer were introduced to the 5’end of the reverse gene primer (DNA
technology).
Forward primers and reverse primers (Invitrogen) for genes B, D and E contain restriction endonuclease recognition sites. At the 3’ end of the forward primer a ClaI restriction site was introduced, while a NotI restriction site was introduced at the 5’ end of the revers primer. Because T4 ligase (Fermentas) was used for cloning, no PCR
amplification of the expression vector, pGAPZαC, was necessary. The pGAPZαC expression vector has many restrictions sites, whereof two of them are ClaI and NotI restriction sites.
INFUSION CLONING PROCEDURE
Cloning of the genes A, B and C to expression vector pGAPZαA was done by using
infusion cloning technique and the reagents and protocol was supplied by Clontech. PCR gen amplification was performed on the gene template and pGAPZαA with suitable primers to generate the desired fragments, followed by ethanol precipitation of the PCR sample. The pellet was digested following the protocol with enzyme DpnI (Fermentas) in 37°C over night. The digested sample was run on an agarose gel and the band with the right size of the target gene was cut out from the gel. Gel extraction was performed on the gel piece using a gel extraction kit from Fermentas. The concentration of the gen in the gel extraction sample was measured using Nano-‐drop (Thermo Scientific). Infusion cloning and transformation to Top10 E. coli (Invitrogen) was performed according to the protocol provided by Clontech. Spin-‐column purification, Step 5 in the protocol was not performed during the procedures. Transformed Top10 E. coli were diluted 10 times and 1/10 of the volume was plated on autoclaved LB agarose plates with 25μg/ml Zeocin (Invitrogen). The plates were incubated at 37° C for 24 hours and after 24 hours visible colonies were picked to a new plate and used for colony PCR-‐screening using
Gotaqgreen (Promega). Positive colonies were incubated in 10ml autoclaved LB (0.5%
