Biotechnology: The Basics PDF 2021

Biotechnology: The basics Mark Carlile Dale 1.06 [email protected] Biotechnology Intro | Mark Carlile 1 Overview: This week we will cover: The basics of biotechnology – the aims A little historical perspectives A look at the current (and future) trends in biotechnological research Biotec...

Biotechnology: The basics Mark Carlile Dale 1.06 [email protected] Biotechnology Intro | Mark Carlile 1 Overview: This week we will cover: The basics of biotechnology – the aims A little historical perspectives A look at the current (and future) trends in biotechnological research Biotechnology Intro | Mark Carlile 2 Associated essay questions 1. Discuss the uses of biotechnology and how it has changed over the past 100 years. 2. Discuss the various expression systems available to biotechnologists and what drives the choice of an expression system/expression vector Biotechnology Intro | Mark Carlile 3 What is biotechnology The use of biological systems to generate useful products More specifically: It is the exploitation of biological processes for industrial and other purposes, especially the genetic manipulation of microorganisms for the production of antibiotics, hormones and recombinant proteins Biotechnology Intro | Mark Carlile At its core Biopharm Science is Biotechnology 4 Holistic aims of modern biotechnology Biotechnology Intro | Mark Carlile 5 Historical biotechnology Biotechnology is probably ~ 6000 years old: 6000 BC : Egyptians used yeast to leaven bread and make alcohol via fermentation 1521 : Aztecs cultivated and harvested algae as a food source 1869 : Johan Meischer isolates DNA from WBCs 1928 : Alexander Fleming : Discovery of penicillin from a bread mold 1953 : Watson and Crick : DNA structure elucidation 1973 : Genentech produce human insulin in E.coli Biotechnology Intro | Mark Carlile 6 Future focus of biotechnology Biotechnology is now embracing all of the current recombinant DNA and cellular engineering technologies Examples: Transgenic animals Gene editing Multigene cassettes – multiple products/synthetic pathways (Biotransformation) Tissue regeneration Protein production and protein engineering Gene therapy Biotechnology Intro | Mark Carlile 7 Exogenous gene expression Expression of non-native genes in an organism Expression is driven via host cell expression “machinery” Use of native and non-native promoters Expression can be via plasmids or via integration into the host genome Biotechnology Intro | Mark Carlile 8 Expression vectors F1 phage origin : viral packaging Reporter gene Antibiotic resistance Protein tag – assay/purification Promoter (T7 phage) lacI gene Origin or replication Various restriction enzyme sites are present Biotechnology Intro | Mark Carlile 9 F1 phage origin : viral packaging Expression vectors Reporter gene Antibiotic resistance Protein tag – assay/purification Important sequences in the pET32a vector Promoter (T7 phage) lacI gene Promoter. Regulatory sequences Origin or replication Various restriction enzyme sites are present Cleavage sites Biotechnology Intro | Mark Carlile 10 Nucleic acids: Manipulation: Cutting Restriction enzymes DNA can be cut in a sequence-specific manner by restriction endonucleases. These enzymes bind to specific DNA sequences and break phosphodiester bond in both strands to generate sticky or blunt ends Compatible sticky ends will anneal because of complementary base pairing Frequency in cutting dependent on the length of recognition sequence: 4 bp recognition seq cut ~ every 256 bp 6 bp recognition seq cut ~ every 4096 bp 8 bp recognition seq cut ~ every 65,536 bp Dr Mark Carlile | DNA/RNA Protein in the Lab 11 Cloning into an expression vector When cloning any gene : moving it from one DNA sequence to another – you need to have restriction sites flanking (at the ends of) the gene YGOI = Your gene of interest Orientation is important Cut both DNA molecules: donor and receiver with the same restriction enzymes usually use different Res-Enzyme to stop re-ligation and to preserve orientation of the insert Gel purify the two fragments : Gel electrophoresis – band extraction – DNA purification Join the fragments together using DNA Ligase (T4 DNA ligase) usually use 3X as much insert as receiver vector : e.g 3 ug insert : 1 ug receiver plasmid Verify cloning via gel electrophoresis Biotechnology Intro | Mark Carlile 12 1000 900 800 700 500 400 300 200 100 - Product digest Purified product Ligation RXN Insert cut Vector cut Insert MW Vector Gel electrophoresis 1000 1000 700 700 400 700 300 300 100 Gel electrophoresis is used to verify sizes and to separate/purify fragments Purified product is used for transforming the cell (host) The product (plasmid+insert) can be digested with REs (same/different) to verify that the ligation has worked Biotechnology Intro | Mark Carlile 13 Expression vectors: Eukaryotes Eukaryotic expression vectors are constructed and amplified (get more DNA) in microbial systems The basic components are the same Most use viral promoters : CMV/SV40 for strong expression More specific promoters will be used for lower expression levels (control) or for cell-specific expression patterns (mammalian expression) Biotechnology Intro | Mark Carlile 14 Host cells: prokaryotic For most biotechnology processes E.coli can/is used Specific mutations are used in the host cell to enhance the productivity, stability or complexity of the expressed product(s) For example: Strong expression : T7 promoter Stabilization of the mRNA Stabilization of the plasmid : T7 lysozyme (breaks down any T7 Polymerase prior to induction Disulfide bond formation and shuffling Biotechnology Intro | Mark Carlile 15 Expression systems Expression Host Your expression system will generate your protein product Mammalian Cells Correctly folded and fully post-transcriptionally modified Transported to the Culture media Available for purification Bacterial / Yeast Cells You have to choose your expression format at the start of your cloning work. Intracellular Expression Soluble Insoluble Cell Breakage Required Extracellular Soluble Folded (PT Modified?) Expression Available for Purification and modification (if required) Dr Mark Carlile : BPS302 Week 15 (In-class slides) 16 Expression vectors and the E.coli lac operon Dr Mark Carlile : Biologics Slide 17 Recombinant vectors Controllable Promoter (inducible) Origin of replication (ori) Selection gene (ampr) Transcriptional control elements Ribosome binding site (ATG) Targeting Sequence (N-term) pBPS302 plasmid Gene of interest (CDS) Targeting Sequence (C-term) Termination sequence Expression cassette PlacI lacI Plac MKKTAIAIAVALAGFATVAQA (ompA Signal Sequence) Olac lac Z lac Y lac A Example: the lac operon Dr Mark Carlile : Biologics Slide 18 The lac operon lacZYA transcription cassette Polycistronic mRNA PlacI lacI Plac Olac lac Z lac Y lac A lacZ : β-galactrosidase lacY : galactoside permease lacA : thiogalactoside transacetylase Wild-type molecular switch lac repressor (Homotetramer) lac Operator Plac Wild-type molecular switch Binging of lacI to the operator is cooperative Binding of lacI to Olac promotes the binding of RNA polymerase Low level expression of lacZYA 28 bp 28 bp + RNA Poly’ase binding Plac lac A Dr Mark Carlile : Biologics lac Y lac Z Slide 19 The lac operon + RNA Poly’ase binding Plac Wild-type molecular switch Binging of lacI to the operator is cooperative Binding of lacI to Olac promotes the binding of RNA polymerase Low level expression of lacZYA Un-induced lac A lac Y lac Z + RNA Poly’ase β-galactosidase Plac Galactoside permease (lacY) allows lactose uptake from media β-galactosidase (lacZ) converts lactose to: - Glucose + Galactose (main reaction) - Allolactose (side reaction) Allolactose binds to lacI and changes its conformation lacI repressor tetramer dissociates from the operator (Olac) Transcription of lacZYA genes Induced lac A lac Y lac Z RNA Poly’ase Plac Induced RNA Poly’ase lac A mRNA lac Y lac Z Transcription Dr Mark Carlile : Biologics Slide 20 Use of the lac operon control elements for heterologous protein expression RNA Poly’ase P Binging of lacI to the operator is cooperative Binding of lacI to Olac promotes the binding of RNA polymerase Low level expression of lacZYA + binding lac lac A lac Y lac Z Gene of Interest lac A Remove the lacZYA genes – restriction cleavage Clone-in you gene of interest Control by IPTG induction lac Z Molecular Biology (Restriction enzyme cleavage) Molecular Biology (Cloning-in) lac Y RNA Poly’ase P + binding lac Gene of Interest This promoter-expression-cassette works but is prone to leakage (switched on in the absence of lactose) Need a more tightly controlled promoter-expression-cassette Leaky expression leads to plasmid instability – plasmid loss Dr Mark Carlile : Biologics Slide 21 Use of the lac operon control elements for heterologous protein expression: T7 RNA Polymerase T7 Poly’ase T7 Promoter Gene of Interest The T7 promoter is taken from bacteriophage T7 (not found in wild type E.coli cells) T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction. The T7 polymerase is expressed using the E.coli RNA Polymerase via the lacUV5 promoter λDE3 Lysogen LacUV5 promoter (lacI binding site) + T7 polymerase gene sequence T7 Poly’ase Inhibition of T7 polymerase expression PlacI and lacI (Lac Repressor) E.Coli chromosome (+ λDE3 Lysogen and lacI cassette) Dr Mark Carlile : Biologics Slide 22 Use of the lac operon control elements for heterologous protein expression: T7 RNA Polymerase λDE3 Lysogen LacUV5 promoter: lacI binding site T7 polymerase gene sequence RNA Poly’ase Plac + binding T7 Polymerase PlacI and lacI (Lac Repressor) se mRNA T7 Polymera E.Coli chromosome (+ λDE3 Lysogen and lacI cassette) Translation T7 RNA Polymerase protein Inducible expression RNA Poly’ase PlacI lacI or m lac Repress RNA Translation lac repressor (lacI) protein Low level constitutive expression Dr Mark Carlile : Biologics Slide 23 Use of the lac operon control elements for heterologous protein expression: T7 RNA Polymerase Un-induced state RNA Poly’ase PlacI E.Coli chromosome lacI or m lac Repress RNA lac repressor (lacI) protein Translation Low level constitutive expression RNA Poly’ase No T7 Polymerase available + lac I bound to operator + binding Plac T7 Promoter T7 Polymerase se mRNA T7 Polymera Gene of Interest No expression of gene of interest X Translation E.Coli chromosome T7 RNA Polymerase protein No Expression of the T7 Polymerase Dr Mark Carlile : Biologics Slide 24 Use of the lac operon control elements for heterologous protein expression: T7 RNA Polymerase RNA Poly’ase PlacI E.Coli chromosome lacI or m lac Repress RNA Induced: IPTG (Lactose analogue) lac repressor (lacI) protein Translation Low level constitutive expression T7 Polymerase expressed + lac I dissociated from operator RNA Poly’ase Plac T7 Promoter T7 Polymerase se mRNA T7 Polymera Gene of Interest Expression of gene of interest Translation E.Coli chromosome T7 RNA Polymerase protein Expression of the T7 Polymerase Dr Mark Carlile : Biologics Slide 25 Use of the lac operon control elements for heterologous protein expression: Overview Gene Cassette LacI Cassette T7 Polymerase Gene of interest Cassette Location E.Coli Chromosome E.Coli Chromosome Plasmid vector Promoter RNA polymerase RNA Polymerase T7 Polymerase Operator - LacI controlled LacI controlled Un-Induced Constitutive - ON OFF OFF Induced Constitutive - ON ON ON Dr Mark Carlile : Biologics Slide 26 Expression vectors: Chinese hamster ovary cell and mammalian expression systems Dr Mark Carlile : Biologics Slide 27 Mammalian expression system Production of recombinant proteins in mammalian cells has allowed the manufacture of a number of large, complex glycosylated polypeptides for clinical applications The mammalian host cell of choice is the Chinese Hamster Overy (CHO) cell CHO cells are adherent but can be grown in stirred bioreactors as a suspension (Protein Expression) The cells require proline in the medium for growth. CHO cells are maintained suspended in culture by revolving culture continuously at approximately 50 RPM. CHO cells should be cultured in DMEM modified with 10% FBS. If cells are not doubling every 14-17 hours, supplement the medium with 1-2% FCS. DMEM - Dulbecco's Modified Eagle Medium FBS – Fetal bovine serum FCS – Fetal calf-serum Can take a while to generate a stable line (expressing) Once the cell line is growing Dr Mark Carlile : Biologics Slide 28 Mammalian expression system: The CMV Promoter An example mammalian expression promoter: The strong human cytomegalovirus (hCMV) promoter regulatory region drives constitutive protein expression levels as high as 50 mg/L. For less potent cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive cells. CMV vectors contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the β-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the f1 origin. Vectors containing the pre-pro-trypsin leader (PPT) sequence direct secretion of fusion proteins into the culture medium for purification using antibodies, resins, and plates. The CMV Promoter: cis acting sites Species Unique Seq Enhancer Seq -550 Promoter -50 Leader Seq +1 Dr Mark Carlile : Biologics CDS +100 Slide 29 Mammalian expression system: The CMV Promoter Cellular Differentiation Ca++ Heat shock Stress TNF-α Serum cAMP PKC IE1 PKA CRE B CCATTGACGTCAATGG CRE Unique Seq ELKSRF 1 CCATATATGG--TTCCG SRE ETS Enhancer Seq CREB: cAMP Response element binding NFκB GGGACTTTCC NF-κB Promoter Leader Seq CDS Complex regulatory pathways CMV is a herpes virus that infects most cell types and establishes latency in leukocytes. The control of the CMV promoter involves many interconnected cellular pathways that both up- and downregulate expression The enhancer sequence is the primary control point Industrial users of this promoter have modified the enhancer sequence and its regulation to their own benefit - Proprietary /Patents Dr Mark Carlile : Biologics Slide 30 Mammalian expression system: The CMV Promoter Unique Seq Enhancer Seq Promoter Leader Seq Advantages CDS Disadvantages Relatively high level of gene expression in differentiated cells (CHO) Low level of expression in un-differentiated cell types (stem cells) Strong preference of the CMV enhancer sequence for cellular signals and transcription factors Strong preference of the CMV enhancer sequence for cellular signals and transcription factors – very tight control of growth conditions is required. Down-regulation is always a concern The enhancer is modulated by a wide variety of chemical/biological mediators The enhancer is strongly unregulated by serum – but there is batch-to-batch variability Transient expression is very strong Stable integration into the genome leads to down-regulation of the promoter – methylation/de-acetylation Dr Mark Carlile : Biologics Slide 31 Mammalian expression system: Inducible promoters Stable inducible mammalian expression vectors are the “holy-grail” for industrial recombinant protein expression - Humanised product from a robust/stable cell-line that can be controllably induced and doesn’t induce any pleiotropic effects (off-target) Some inducible systems used to varying effect are: - Temperature shift responsive elements : 37 – 42 oC (Heat shock promoter) - Heavy metal induction : metallothionine promoter – responsive to zinc, copper, cadmium - Steroid responsive promoters: induction with glucocorticoids The major disadvantages of these expression systems is - the off-target effects due to the generic induction strategies - A high basal (un-induced) expression level leads to a negative regulation upon induction through stress response mechanisms Temperature shift, Zn2+/Cu2+, Steriods Control Seq Promoter Leader Seq Dr Mark Carlile : Biologics CDS Slide 32 Expression host overview Biotechnology Intro | Mark Carlile 33 Making a COVID-19 Vaccine Restriction enzymes SARS-CoV Spike Protein gene SARS-CoV Spike Protein gene Ligase enzymes SARS-CoV Spike Protein gene Transformation SARS-CoV Spike Protein gene Selection of clones Biotechnology Intro | Mark Carlile 34 Making a COVID-19 Vaccine Cell breakage (Homogenisation) Shake flask Production fermenter Cell Harvest (Centrifugation) x3 Final product Formulation Biotechnology Intro | Mark Carlile Chromatographic separation 35 Summary: This week we have covered: The very basics of biotechnology: what it is and what it does We have looked at basic cloning protocols Looked at the different expression systems that are commonly used in biotechnology/bioporcessing Biotechnology Intro | Mark Carlile 36