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Mary is the scientific consultant of a firm of investors interested in biotechnology. She is asked to evaluate the research plan of a small company producing engineered variants of the protease subtilisin from
Bacillus subtilis
used in detergents. The company plans to use
Escherichia coli
as the host for the expression of these variants. The investors Mary is representing ask her to prepare a report evaluating the feasibility of this approach.
Production of the extracellular domains of tissue factor (F3) from Mus Musculus (mouse) using recombinant protein technology for purpose of crystallization. Group 4 Production of the extracellular domains of tissue factor (F3) from Mus Musculus (mouse) using recombinant protein technology for purpose of crystallization. P a g e | 1 GROUP 4 PROBLEM BASED LEARNING Objectives To produce the extracellular domains of Mus musculus (mouse) tissue factor (F3) recombinant protein for the purpose of analysing the structure via crystallisation. Introduction F3 is a clotting factor responsible for the initiation of blood coagulation cascades. F3 also functions as the receptor for coagulation factor VIIa, which when in complex catalyses the proteolytic activation of coagulation factor IX and X (NCBI, 2016). F3 is a membrane bound glycoprotein, consisting of extracellular, transmembrane and cytoplasmic domains. Within the extracellular domain two disulphide bonds play an important role in forming the specific receptor structure required to complex with factor IX and X (Signal Peptide Website, 2010). Crystallisation shall be used to investigate the arrangement of F3, whereby the information received from an X-ray scatter is used to derive the structure. To obtain reliable results, a protein of extremely high purity and of the highest concentration without causing precipitation and aggregation (2-50mg/ml) is required (Dessau & Modis, 2011). These requirements and those of a postdoc shall dictate the method used. P a g e | 2 GROUP 4 PROBLEM BASED LEARNING Workflow Diagram Mouse Tissue Factor (F3) •Using only the extracellular domain •Wild Type •Obtained by gene synthesis Obtain DNA •His-tag attached which can be removed with a thrombin cleavage site •Will add tag to C terminus •Will use α-mating factor as a secretory signal leader Create Expression Clone •Assembled In Escherichia coli Express the protein •Expression host is Pichia pastoris •Conditions required: continuously stirred bioreactor, dissolved O2 content above 20%, temperature of 30oC and pH 5 Selection •Plasmid contains Zeocin resistance •Clonal selection: ELISA to measure concentration of protein Purify the Protein •Cleavage of N-linked glycosylation sites •Nickel immobilised metal affinity chromatography •Cleave thrombin site to remove his-tag •SDS page •Ion exchange chromatography •Gel filtration P a g e | 3 GROUP 4 PROBLEM BASED LEARNING Experimental design Obtain the DNA Protein sequence overview The final protein sequence is depicted in Figure 1. Sequence elements Promoter The experiment has been designed to incorporate the GAP promoter (PGAP), which resulted in the use of the pGAPZ expression vector obtained from Invitrogen. pGAPZ is formed by replacing the methanol-regulated alcohol oxidase 1 (AOX1) promoter (PAOX1) with the constitutive PGAP in the backbone of the pPICZ vector (Thermo Fisher Scientific, n.d.). An 86 amino acid alpha-mating factor signal peptide at the N-terminus of the extracellular domain of the F3 protein. MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLDKRAGIPEKAFNLTWISTDFKTILEWQP KPTNYTYTVQISDRSRNWKNKCFSTTDTECDLTDEIVKDVTWAYEAKVLSVPR RNSVHGDGDQLVIHGEEPPFTNAPKFLPYRDTNLGQPVIQQFEQDGRKLNVV VKDSLTLVRKNGTFLTLRQVFGKDLGYIITYRKGSSTGKKTNITNTNEFSIDVEE GVSYCFFVQAMIFSRKTNQNSPGSSTVCTEQWKSFLGELVPRGSHHHHHH Extracellular domain of F3 A His tag at the C- terminus of the protein A thrombin cleavage site Figure 1: Annotated final protein sequence that will be expressed for crystallisation, containing the alpha mating factor signal peptide Sna16 \l 2057 at the N-terminus, the extracellular domain of the F3 protein Tis16 \l 2057 and a thrombin cleavage site and His tag at the C-terminus (SnapGene, 2016) (NCBI, 2016). Commented [JM1]: I rearranged your labels that were in the wrong place. But excellent you got all the correct elements, you removed the secretory leader from F3 and the transmembrane and cytoplasmic tail. P a g e | 4 GROUP 4 PROBLEM BASED LEARNING Alpha-mating factor Alpha-mating factor is an 86 amino acid signal peptide from Saccharomyces cerevisiae. It has been placed immediately upstream of the F3 sequence in the open reading frame of the expression cassette (Lin-Cereghino, et al., 2013). This will ensure that the nascent peptide is directed into the endoplasmic reticulum for incorporation into the secretory pathway. The alpha-mating factor sequence is split into a pre-sequence of 19 amino acids, followed by a pro-sequence of 67 amino acids (Lin-Cereghino, et al., 2013). The pro-sequence contains three N-linked glycosylation sites and a dibasic Kex2 endopeptidase processing site. Processing of the signal peptide occurs in three different stages, with the first consisting of the pre-signal being removed in the endoplasmic reticulum. Subsequently a Kex2 endopeptidase cleaves the pro-sequence and then in the gGolgi a Ste13 protein cleaves the Glu-Arg repeats, completing the processing of the signal peptide. The pre-sequence is thought to be responsible for the translocation of the signal peptide and, therefore, the F3 protein of interest to the endoplasmic reticulum (Lin-Cereghino, et al., 2013). This sequence is cleaved off naturally in the cell before crystallisation. His Tag incorporating thrombin cleavage site A polyhistidine-tag (His-tag) consisting of 6 histidine codons is attached to the coding sequence of the thrombin consensus sequence. The tag, attached to the C-terminus, can then be cleaved after purification using the protease thrombin. 3’ UTR A 3’-UTR from the PAOX1 gene is added to the 3’ end of the transcription cassette in order to increase the stability and half-life of the mRNA (Shalgi, et al., 2005). Create expression clone The expression cassette (Figure 2) is incorporated into the pGAPZ plasmid multiple cloning site. The restriction enzymes XhoI or NotI could be used for this. The plasmid is then introduced into Escherichia coli where it is amplified to obtain a larger quantity. The plasmids are then isolated and transformed into the CBS7435 ku70 strain of Pichia pastoris. Commented [JM2]: Very nice research Commented [JM3]: Minor detail but include the thrombin cleavage site LVPR/GS so you will have an additional sequence of LVPR on the C-terminus. Commented [JM4]: Why this particular strain what does it offer? You have all the elements in your work flow but why not describe them here. Transformation by electroporation, selection on zeocin. Clonal selection for high producer. P a g e | 5 GROUP 4 PROBLEM BASED LEARNING Express the protein The expression host is a strain of yeast called Pichia pastoris. It should be cultured in a continuously stirred tank bioreactor with a dissolved oxygen concentration above 20%. The temperature should be kept at 30oC and the media should be at pH5 (Darby, et al., 2012). The broth will also contain the protease inhibitors pepstatin, E-64, EDTA, and pefabloc to minimise degradation of the protein by endogenous secreted proteases (Shi, et al., 2003). The N-linked glycosylation sites will be removed using endoglycosidase H as glycosylation interferes with crystallisation (Cregg, et al., 2000). If proteins start to misfold it is possible to use a strain of yeast that overexpresses folding enzymes and chaperones (Mattanovich, et al., 2011). Transcription cassette The transcription cassette that will be inserted into the pGAPZ plasmid is depicted in Figure 2. Figure 2: The transcription cassette that will be expressed, containing a GAP promoter, the alpha-mating factor signal peptide, the extracellular domain of the F3 protein, a thrombin cleavage site, His tag and 3’-UTR from the AOX1 gene. Commented [JM5]: I now realise this whole section is in the wrong place! Move to after selection. Formatted: Font: Italic Commented [JM6]: Why? Commented [JM7]: In the wrong place?You could have introduced sequence changes in your construct to destroy the N-linked glycosylation consensus sequence NXS/T. Replacing S or T with Alanine would be conservative. Commented [JM8]: No ribosome binding site? Bullet list of other important elements might have included: Origin replication for Ecoli Amp resistance for selection in Ecoli Zeocin resistance expression cassette for selection in Ppastoris. P a g e | 6 GROUP 4 PROBLEM BASED LEARNING Selection The yeast cells which have taken up the plasmid will be selected for by zeocin resistance as the plasmid used contains a zeocin marker. Clonal selection is carried out in a 96-well plate, whereby the cells are diluted to the concentration where there is only a single cell in each well. An ELISA will be used to screen for cells that secrete the protein of interest in sufficient quantities. Purification Initially, nickel immobilised metal affinity chromatography (IMAC) will be used to purify the protein. The nickel on the column will bind the His-tag and whilst still bound to the column, thrombin is used to cleave the sequence between the His-tag and the protein, releasing the latter from the column. The success of this will be confirmed via SDS-PAGE. To further purify the protein, ion exchange chromatography (IEX) and gel-filtration chromatography will be used in succession. P a g e | 7 GROUP 4 PROBLEM BASED LEARNING Discussion Protein Sequence Disulphide/glycosylation It is important for protein crystallisation that the two disulphide bonds in the extracellular domain form correctly because failure to do so may result in an incorrect protein structure. Additionally, N-linked glycosylation is not desirable when undertaking protein crystallisation due to the fact that carbohydrates can interfere with this process. On the other hand, glycosylation can help the protein form its proper structure (Chang, et al., 2007), therefore a method whereby glycosylation occurs but can then be cleaved off is desirable. UTRs UTRs can be placed at either end of the transcription cassette in order to influence expression. 5’-UTRs usually control the rate of expression by modulating the efficiency of transcriptiontranslation, with the secondary structure regulating the progression of the ribosome along the mRNA (Staley, et al., 2012). 3’-UTRs have been shown to increase the half- life of mRNA, leading to increased expression. In the P. pastoris expression system, the 5’-UTR of the PAOX1 gene is commonly used as it increases levels of protein expression. However, because PGAP is being used to drive expression in this system, the uncharacterised effects of the AOX1 5’-UTR on the PGAP makes it unwise to use in this case due to potential problems in protein expression. However, use of the AOX1 3’-UTR is unlikely to be a concern, meaning it should increase protein expression. Alternatively, the M8 and M11 stabilising motifs identified in S. cerevisiae, could be used to increase mRNA half-life (Shalgi, et al., 2005). Additionally, as many motifs are highly conserved across species, use in