Figure 41: the Recombinant Serglycin-His-Flag

4Results

4Results

Serglycin is a proteoglycan with eight repetitive glycosaminoglycan attachment sites. In most cells studied, these attachment sites do preferentially contain chondroitin sulphate (Kolset et al., 1996), with the exception of mast cells where the core protein contains a mixture of chondroitin sulphate and heparin (Lidholt et al., 1996). Due to protease activity, most of the core protein is often degraded before the proteoglycan is secreted. Only a few amino acids remain on each side of the glycosaminoglycan attachment region. The short core protein, and the shielding by the glycosaminoglycan chains, makes the proteoglycan less immunogenic. Detection of serglycin molecules is thus difficult.

In this post-graduate thesis, the issue was to study the secretion of this proteoglycan, in both endothelial cells and epithelial cells. To facilitate isolation and detection of the proteoglycan, it was decided to make a recombinant serglycin by coupling a myc-His-flag to the C-terminal end of the serglycin core protein. The His-flag favours purification by mediating binding to transition metal ions that are coupled to for instance a matrix. Detection of the proteoglycan is facilitated by the His-flag and the myc-epitope, which both can be recognised by antibodies. Figure 41, shows the recombinant serglycin-His-flag.

Figure 41: The recombinant serglycin-His-Flag.

The region with the attached glycosaminoglycan chains represents the mature serglycin core protein, and the dotted area the extra protein sequence containing the myc-epitope and the C-terminal 6xHis-epitope.

4.1Molecular cloning of serglycin-His-flag

4.1.1pcDNA3.1(-)/Myc-His vector

The pcDNA3.1(-)/Myc-His vector (5.5 kb) was used to insert serglycin cDNA when cloning the serglycin-His-flag. It contains a cytomegalovirus immediate-early (CMV) promoter that provides high expression of the recombinant protein in a wide range of mammalian cells, and a bovine growth hormone (BGH) polyadenylation signal that terminates the His-flag gene with a polyA sequence. The polyA tail stabilises the mRNA transcript by making it less exposed for degradation by RNAses in the cytoplasm. The vector also contains a cDNA that codes for the neomycin resistance gene, that can be used for selection of stably transfected cells. A plasmid drawing of the pcDNA3.1(-)/Myc-His vector is presented in Figure 42.

Figure 42: The pcDNA3.1(-)/Myc-His vector.

Selection of restriction site for insertion of serglycin cDNA

In the pcDNA3.1(-)/Myc-His vector, the insertion site for the cDNA is located upstream of the region encoding the myc-epitope and the His-Flag. By removal of the stop signal in the inserted cDNA, the translation continues into the vector. Both the myc-epitope and the His-Flag are translated before the translation stops at the stop codon terminating the His-Flag. To get a correct translation of the tail, it is important that the cDNA is inserted in reading frame with the Myc-His. To ensure this, there are three different pcDNA3.1(-)/Myc-His vectors, A, B and C. They have different multiple cloning sites, giving three different reading frames.

When selecting the insertion site for the serglycin cDNA, a special attention was taken to keep most of the multiple cloning site region. By keeping this region, the C-terminal end with the His-flag would end up longer from the dominating negatively charged glycosaminoglycan chains, hopefully keeping them from disturbing the binding of the his-tag to the transitional metal matrix (Figure 46).

It was also of interest to choose restriction sites that give single stranded termini, and which are easily cleavable by common restriction enzymes. Two different restriction sites were chosen to reduce the possibility of religation of the vector, incorrect inserts or incorporation of successive serglycin cDNA. It was chosen to insert the serglycin cDNA by the restriction enzymes XbaI and EcoRI (Figure 43).

Figure 43: Selection of restriction sites in the pcDNA3.1(-)/Myc-His vector.

4.1.2serglycin cDNA

The available serglycin cDNA, containing the open reading frame (ORF) of serglycin (Uhlin-Hansen et al, 1993), was inserted into a pBluescript vector with the restriction sites BamHI and XhoI. These restriction sites did not match with those selected in the pcDNA3.1(-)/Myc-His vector, and could not be used to insert the serglycin cDNA. Therefore, serglycin ORF was amplified by PCR using a PCR upper primer with the restriction site XbaI and a PCR lower primer with the restriction site EcoRI. The primers were selected by using the PC programme Oligo (Medprobe).

As explained previously, removal of the stop sequence in the serglycin cDNA was necessary for translation of the C-terminal myc-His tag. This stop codon was removed by putting the PCR lower primer immediate upstream of the translation stop sequence (TGA) in the serglycin cDNA (Figure 44). When suitable primers were found, the in frame cloning of the myc-His-Flag was rechecked, and found to be maintained with the pcDNA3.1(-)/Myc-His A vector.

Figure 44: Selection of PCR primers and sites

A: Location of the PCR primers used for the exchange of restriction sites, and removal of the stop codon in serglycin cDNA by PCR. B: The polynucleic acid sequence in the PCR primers used.

4.1.3Transcription and translation of the recombinant serglycin-His-flag

Ligation of the PCR amplified serglycin cDNA into the pcDNA3.1(-)/Myc-His A vector results in the recombinant serglycin, from now on named serglycin-His-flag. The new vector is named pcDNA3.1(serglycin)/Myc-His. With the insertion of the serglycin cDNA into the pcDNA3.1(-)/Myc-His vector, the pcDNA3.1(serglycin)/Myc-His vector increased in size from 5,5 to 5,9 kb (Figure 45).

Figure 45: Cloning of the serglycin-His-flag gene.

A pBluescript vector with serglycin cDNA (Uhlin-Hansen et al, 1993) and the pcDNA3.1(-)/Myc-His A vector (InvitroGen) were used to construct the serglycin-His-flag. In the cloning process, the stop codon in the serglycin cDNA was removed with a lower PCR primer, making the reading frame continue into the pcDNA3.1(-)/Myc-His A. This resulted in the hybrid serglycin core protein with a myc-epitope and a C-terminal His-flag. Since the pcDNA3.1(-)/Myc-His A vector lacks a Kozak translation initiation sequence, the ATG codon in the serglycin cDNA was kept and inserted into the vector.

Transcription of the serglycin-His-flag mRNA is determined by the localisation of the putative transcription start signal and the polyadenylation signal (AATAAA) in the vector. After insertion of the serglycin cDNA, the number of base pairs between these two signals is 737. With termination 20 bases behind the polyadenylation signal, the total mRNA transcript becomes approximately 760 bp. In contrast, the human genomic serglycin mRNA is approximately 1,3 kb. The serglycin-His-flag mRNA is shorter than the human serglycin mRNA, since the serglycin cDNA inserted into the pcDNA3.1(-)/Myc-His vector, lacks the 3´and the 5´untranslated ends.

Translation of the serglycin-His-flag gives a core protein of 192 amino acids. The translated serglycin core protein is increased by 38 aa, from 154 aa, by the attachment of the myc-His-flag. Although the attached myc-His-Flag contributes to an increase in the size of the core protein, it does not significantly influence the molecular weight of the proteoglycan. The serglycin core protein has a molecular weight of approximately 10 kDa, but the secreted proteoglycan from U937 cells has a molecular weight of 200 kDa or more.

Figure 46: The pcDNA3.1(serglycin)/Myc-His vector.

A: The new pcDNA3.1(serglycin)/Myc-His vector with all functional genes. B: Transcription and translation of the serglycin-His-flag. The additional protein tail is C-terminal.

4.1.4Construction of the pcDNA3.1(serglycin)/Myc-His vector

The pcDNA3.1(-)/Myc-His vector is a shuttle plasmid, and is thus able to replicate in both prokaryote and eukaryote cells. That simplified the molecular cloning, since the genetic engineering could be done in E. coli. (A prokaryotic cell is preferred to an eukaryotic cell, since bacteria are easier to grow and transform and are better source for plasmid isolation).

The molecular cloning of the pcDNA3.1(-)/Myc-His vector was done according to methods common in most laboratories, and will not be described in details in this chapter. All reactions are performed as described in method chapters 3.1 and 3.2.

The step that may differ from ordinary laboratory practice, is the ligation of the PCR product into the pCRTMII vector before the restriction analysis. After amplification of the serglycin cDNA by the PCR reaction, the PCR products were ligated directly into a linearised pCRTMII vector. This vector contains a single deoxythymidine (dT) in the 3' ends. Since the Taq polymerase used in the PCR reaction adds a single deoxyadenosine (dA) to the 5' ends on the PCR product, the PCR product will ligate efficiently into the vector. The PCR product was inserted into the TA vector to simplify the action of the restriction enzymes in the following step. Restriction enzymes digest DNA poorly if the restriction site is not flanked by DNA, and the restriction sites inserted into the PCR primers, end up only few bases from the two ends in the PCR product.

The cloning procedure is schematically illustrated in Figure 47. The left lane shows the main steps during the cloning, while the right lane shows the methods used in each step. Verification of successful reactions in each step during the molecular cloning is not shown.

Verification of serglycin insert

The last step in the cloning procedure was to verify that serglycin cDNA was successfully inserted into the pcDNA3.1(serglycin)/Myc-His vector. Restriction analysis followed by agarose gel electrophoresis was performed. A successful insertion is proven if the serglycin cDNA can be digested out of the vector within the restriction sites used for insertion.

From the transformation plates obtained in the last step in Figure 47, eight parallel colonies were picked for mini-preparation of plasmid DNA (method 3.2.4). Both undigested and EcoRI and XbaI digested vectors were analysed by agarose gel electrophoresis (method 3.1.4). All picked colonies contained the serglycin cDNA (result not shown).

Two of these colonies were picked for maxi preparation of plasmid DNA (method 3.2.5). The concentration of isolated DNA in each sample was determined by spectrophotometric quantitation at 280 and 260 nm (method 3.1.1) prior to restriction analysis (method 3.1.2). Both undigested and EcoRI and XbaI digested vectors were analysed by agarose gel electrophoresis (method 3.1.4). A parallel maxi-prep isolation of the pcDNA3.1(-)/Myc-His A vector was performed to obtain control material. To be able to see the insert and possible contaminations clearly, 2 g DNA was used per well.

Figure 47: Flow chart describing the methods used to construct the serglycin-His-flag.

The restriction analysis of two parallel pcDNA3.1(serglycin)/Myc-His vectors and the original pcDNA3.1(-)/Myc-His vector is shown in Figure 48. Lanes 3, 5 and 8 contain undigested plasmid, while lanes 2, 4 and 7 contain digested plasmid. The serglycin cDNA fragment is shown in lane 2 and 4, but not in lane 7, as expected. In both plasmid preparations, digestion of the pcDNA3.1/(serglycin)/Myc-His vector resulted in a small fragments with the expected sizes of ~500 kDa, verifying that all selected colonies contained successfully inserted serglycin cDNA. No contaminating DNA was seen in either of the samples.

Figure 48: Restriction analysis of the pcDNA3.1(serglycin)/Myc-His vector

Maxi prep isolated pcDNA3.1(serglycin)/Myc-His vector and the original pcDNA3.1(-)/Myc-His A vector were analysed by agarose gel electrophoresis. 2 l DNA was applied in each lane to be able to see contamination.

The three plasmid preparations shown in Figure 48 were selected for transfection of the endothelial- and epithelial cell lines. To reduce the possibility of transfecting the cells with a mutant pcDNA3.1(serglycin)/Myc-His vector, both the vectors isolated from colony 1 and colony 4 were selected. A mutation could be introduced in the cDNA by the Taq-polymerase used in the PCR reaction, due to the enzymes error rate of 210-4 nucleotides per cycle. A mutation in both products was found very unlikely.

The two pcDNA3.1(serglycin)/Myc-His vectors carrying serglycin were given the prefix number 1 and 4. As a control, the cells were also transfected with the pcDNA3.1(-)/Myc-His vector without insert. This vector was given the prefix 7 (Table 41). If not commented further, all transfection assays were done with these three vectors in parallel. The pcDNA3.1(serglycin)/Myc-His vector isolated from colony 1 was sent to Eurogenetic, Belgium, for control sequencing, and found to code the suspected DNA sequence (Appendix 1).

Prefix number / Description
1 / serglycin insert / PCR product 1
4 / serglycin insert / PCR product 2
7 / no insert / -

Table 41: The vectors selected for transfection.

4.2Transfection of MDCK II cells and endothelial cells

The secretion study of the serglycin-His-flag proteoglycan, requires introduction and expression of the pcDNA3.1(serglycin)/Myc-His vector in the cells to be studied. Introduction of foreign DNA into mammalian cells is performed by transfection. Transfection can be divided into two types, transient transfection and stable transfection. In transient transfection studies, the gene product or biochemical reactions are analysed when the cDNA has entered the nucleus and become expressed, usually few days after transfection. High transfection efficiency is thus important to see the biological effect of the gene. During transient transfection, some cells stably integrate the foreign DNA into the genome. These stably transfected cells can be isolated from the others with a positive selectable marker in the transfection vector.

The four most common transfection methods are calcium phosphate transfection, DEAE-dextran transfection, electroporation and liposome mediated transfection. To choose between them is difficult, since cells do respond differently to the different transfection methods. Cells of different origin also show variation in the transfection efficiency, making it difficult to find the best method to transfect a certain cell line. Consequently, each method must be optimised for each cell line.

The aim of this project was to study the secretion of the serglycin-His-flag proteoglycan in polarised cultured cells. This required stably transfected cell lines, because transient transfected cells would eject the vector before the cell layer was grow confluent. As mentioned above, the stably transfected cells can be isolated from the others by a selectable maker, and the pcDNA3.1(serglycin)/Myc-His vector contains a neomycin resistance gene, that gives resistance to the aminoglycoside geneticin (G-418). To get stably transfected subclones, the transfected cells had to be cultured in medium containing geneticin.

4.2.1Transfection of the MDCK II cell line

Choice of transfection method

A method optimised for stable transfection of the MDCK II cell line already existed at the University of Oslo (Nordeng, T., personal communication; Simonsen, 1997). It was a calcium phosphate transfection method (optimised after Ausubel, et al., 1996), with the use of geneticin as the selectable marker for selection of stably transfected cells.

With the calcium phosphate method, the DNA is introduced to the cells via a DNA precipitate that adheres to the cell surface. A buffer is used to form a calcium phosphate and DNA precipitate that is directly layered onto the cells. Depending on the cell type, up to 10 percent of the cells take up the DNA precipitate through an undetermined mechanism. The calcium phosphate transfection is often followed by DMSO shock to improve transfection efficiency. The method is efficient for both transient and stable transfection of adherent cells. It introduces large amounts of DNA into the cells that pick up the DNA, which increases the possibility that some of it will be stably integrated into the genome.

Transfection

The MDCK II cells were transfected with the calcium phosphate method in co-operation with Tram Thu Vuong, another student in the laboratory (method 3.5.2). The three different DNA constructs 1, 4 and 7 were used (vector number 1 and 4 are both carrying serglycin cDNA, while the vector number 7 is the original pcDNA3.1(-)/Myc-His A vector).

3  105 MDCK II cells were seeded on a 10 cm petri dish in 10 ml complete medium one day prior to transfection. The next day, 20 l DNA for each transfection was suspended onto the cells in the petri dish and incubated for 20 hours. After transfection, the MDCK II cells were shocked with cold DMSO for 1 hour.

Two days after transfection, the cells were splitted 1:100, 1:30, 1:10 and ~9:10, and given selective medium (500 g/ml G-418). After fourteen days with selective medium, stably transfected colonies were picked with cloning rings from 1:100 and 1:30 diluted petri dishes (control cells died after ten days) (method 3.5.4). Eleven colonies were chosen from the dish transfected with construct 1, and given the number 1-1 to 1-11. Five colonies were chosen in the dish transfected with construct 4 (4-2 to 4-6), and three colonies were chosen from the dish transfected with construct 7 (7-2 to 7-4). Each transfected sub-cell line was splitted when confluent, and seeded into bigger wells to increase the cell number. After ten to fifteen days, two or more 75 cm2 flasks were obtained for all subclones.

4.2.2Transfection of endothelial cells

Choice of endothelial cell lines

Very few protocols (covers all written matter) describing transfection of endothelial cells exist, and most of them describes transient transfection (Gille et al., 1996; Roebuck et al., 1995). The main reason for this is the difficulty of transfecting endothelial cells. Two different endothelial cell lines were therefore selected to increase the possibility of obtaining a cell line stably expressing the serglycin-His-flag.