Chapter 8: Expression and Modification of Recombinant Proteins Prokaryotic expression system Eukaryotic expression system Yeast expression Insect expression system Mammalian expression system

References Chapter 11 and 13 (Essentials of Molecular Biology) Chapter 10 (An Introduction to Genetic Engineering)

I. Prokaryotic Expression System Friday, April 21, 2017 I. Prokaryotic Expression System The red boxes represent exons, the blue boxes represent the introns, and the grey boxes represent the 5’ and 3’ UTRs the gene of eucaryotic includes additional sequences that lie within the coding region, The sequences of DNA comprising an interrupted gene are divided into the two categories, • The exons are the sequences represented in the mature RNA. By definition, a gene starts and ends with exons, corresponding to the 5' and 3' ends of the RNA. • The introns are the intervening sequences that are removed when the primary transcript is processed to give the mature RNA. How do you express this gene in bacteria? Insert only the ORF: into an expression vector that contains prokaryotic transcriptional and translational regulatory sequences

Six Step Process Isolation of gene of interest Introduction of gene to expression vector Transformation into host cells Growth of cells through fermentation Isolation & purification of protein Formulation of protein product

Cloning Process Gene of interest is cut out with restriction enzymes (RE) Host plasmid (circular chromosome) is cut with same REs Gene is inserted into plasmid and ligated with ligase New (engineered) plasmid inserted into bacterium (transform)

Fundamentals of Gene Expression Prokaryotic and eukaryotic promoters and translation signals are different...they are not exchangeable You therefore can’t simply put a eukaryotic promoter into bacteria and expect it to function

The genetic code is identical for most bacterial and eukaryotic mRNAs. Processing also presents a problem for bacterial expression of human mRNAs. The eukaryotic ORF must be cloned into the expression vector, not the genomic copy The genetic code is identical for most bacterial and eukaryotic mRNAs. Although the code is the same, expression levels can be affected by codon frequency, which varies between organisms and transcripts Post-translational modifications can be important for protein function Those modifications might not occur in bacteria. The solution…try expressing in a eukaryotic expression system (viral, baculovirus, yeast)

Prokaryotic Expression Pros Convenient/Easy Produce and purify protein using the least expensive and easiest reagents and equipment Best for large scale production of protein Cons lack many of the immunogenic properties 3D “Native” conformation Lack PTMs (Post translational modifications) needed for specific activity

Which Vector? Tags标签 Promoters arabinose systems (pBAD), phage T7 (pET), Trc/Tac promoters, λ PL or PR Tags标签 His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coil/K-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags

Gene ( with / without stop codon ) Fusion Protein Gene ( with / without stop codon ) T7 Promoter RBS ATG TAG lac Operator peptide tag: V5 / poly-(his) mutiple cloning site (mcs) peptide tag: V5 / poly-(his)

A Generic Vector

II. Eukaryotic Expression System Yeast expression Insect expression system Mammalian expression system

1. Yeast Expression 􀂄 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous proteins Often comfortable with genetic manipulation Exhibit near-native conformation, PTMs, processing Well-defined secretory pathways for extracellular export of the recombinant gene product Usually safe to use Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eukaryote proteins

Yeast Expression Vector Comments for pPIC9K: 9276 nucleotides 5´ AOX1 promoter fragment: bases 1-948 5´ AOX1 primer site: bases 855-875 a-Factor secretion signal(s): bases 949-1218 a-Factor primer site: bases 1152-1172 Multiple Cloning Site: bases 1216-1241 3´ AOX1 primer site: bases 1327-1347 3´ AOX1 transcription termination (TT): bases 1253-1586 HIS4 ORF: bases 4514-1980 Kanamycin resistance gene: bases 5743-4928 3´ AOX1 fragment: bases 6122-6879 pBR322 origin: bases 7961-7288 Ampicillin resistance gene: bases 8966-8106 Pichia pastoris

Yeast are single celled eukaryotes Behave like bacteria, but have key advantages of eukaryotes P. pastoris is a methylotrophic甲醇为营养的yeast that can use methanol甲醇as its sole carbon source (using alcohol oxidase) Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)

Cloning in Yeast Cells Uses a special plasmid that works both in E.coli and Yeast Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isn’t circular) Double cross-over recombination event occurs to cause the gene of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be Now gene of interest is under control of the powerful AOX promoter

Cloning 3 1 2 4

2. Insect Expression Pros Cons Produce proteins that has PTMs similar to mammalian systems Often properly folded and functional Ideal for producing moderate to high levels of eukaryotic proteins for structure-function assays Cons Expensive Sometimes proteins are not correctly folded Often not stable

Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs) Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae) Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell

Baculovirus (AcMNPV) Cloning Process 5’ 3’ Transfer vector Polyhedrin gene x Cloned gene Cloned gene 5’ 3’ AcMNPV DNA Recombinant AcMNPV DNA

Baculovirus

BacVirus Expression System pIEx™Insect Cell Expression Plasmids (Novagen) Bac-N-Blue™Baculovirus Expression System (Invitrogen)

3. Mammalian Expression Pros Produce protein in the most native and active form Have required PTM machinery to produce active and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant proteins Limitations on the mechanisms of protein expression induction Almost always have over expression

HeLa cells in culture

HeLa cells from the Nikon microscope web site HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s. They have been utilized for many purposes, including the development of a polio vaccine, the pursuit of a cure for diseases such as leukemia and cancer, and the study of the cellular effects of drugs and radiation.

JONATHON PINES REGULATION OF MITOSIS IN MAMMALIAN CELLS HeLa Human cells JONATHON PINES REGULATION OF MITOSIS IN MAMMALIAN CELLS Mitotic HeLa cell stained with anti-Cks1 (red), anti-tubulin (yellow) and DAPI (blue)

HeLa cells as you will see them Expression of (A) b-galactosidase and B green fluorescent protein in HeLa cells. Cells were transfected in 6-well plates. Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection.

Expression System Selection Choice depends on size and character of protein Large proteins (>100 kD)? Choose eukaryote Small proteins (<30 kD)? Choose prokaryote Glycosylation essential? Choose baculovirusor杆病毒 mammalian cell culture High yields, low cost? Choose E. coli Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote

Which Vector Must be compatible with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells) Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site

Key Parts to a Vector Origin of replication (ORI) – DNA sequence for DNA polymerase to replicate the plasmid Selectable marker (Amp or Tet) – a gene, when expressed on plasmid will allow host cells to survive Inducible promoter – Short DNA sequence which enhances expression of adjacent gene Multi-cloning site (MCS) – Short DNA sequence that contains many restriction enzyme sites

Mammalian Expression Gene initially cloned and plasmid propagated in bacterial cells Mammalian cells transformed by electroporation (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression & integration of DHFR and the gene of interest

Methotrexate (MTX) Selection Gene of interest DHFR Transfect dfhr- cells Grow in Nucleoside Free medium Culture a Colony of cells Grow in 0.05 uM Mtx Culture a Colony of cells

Methotrexate (MTX) Selection Grow in 0.25 uM Mtx Culture a Colony of cells Grow in 5.0 uM Mtx Culture a Colony of cells Foreign gene expressed in high level in CHO cells

Mammalian Expression Systems Comments for pcDNA™4/TO : 5078 nucleotides CMV promoter: bases 232-958 TATA box: bases 804-810 Tetracycline operator (2X TetO2) sequences: bases 820-859 CMV forward priming site: bases 769-789 Multiple cloning site: bases 967-1077 BGH reverse priming site: bases 1089-1106 BGH polyadenylation sequence: bases 1095-1319 f1 origin: bases 1365-1793 SV40 promoter and origin: bases 1803-2143 EM-7 promoter: bases 2183-2249 Zeocin™ resistance gene: bases 2250-2624 SV40 early polyadenylation sequence: bases 2754-2884 pUC origin: bases 3267-3937 bla promoter: bases 4937-5041 (complementary strand) Ampicillin (bla) resistance gene: bases 4082-4942 (complementary strand)

Mammalian Cell-line Expression Sometimes required for difficult-to-express proteins or for “complete authenticity” (matching glycosylation and sequence) Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line Vectors usually use SV-40 virus, CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene

Summary Friday, April 21, 2017 The mechanism of regulation of gene expression in prokaryotes is greatly different from eukaryotes Regulation of gene expression in prokaryotes is mainly for environmental adaptation Regulation of gene expression in eukaryotes is for cell growth, differentiation and development There are four recombinant protein expression systems: bacteria (E.coli), yeast, insect and mammalian cells. Choose by purpose