Chapter 8 Expression and Modification of Recombinant Proteins

Chapter 8: Expression and Modification of Recombinant Proteins Prokaryotic expression system II. Eukaryotic expression system I. Yeast expression 2. Insect expression system 3. Mammalian expression system 1. Molecular Biology

References Chapter 11 and 13 (Essentials of Molecular Biology) 2. Chapter 10 (An Introduction to Genetic Engineering) 1. Molecular Biology

I. Prokaryotic Expression System How do you express this gene in bacteria? Insert only the ORF: The red boxes represent exons, the blue boxes represent the introns, and the grey boxes represent the 5’ and 3’ UTRs into an expression vector that contains prokaryotic transcriptional and translational regulatory sequences 2/27/2021 Molecular Biology 3

Six Step Process ① ② ③ ④ ⑤ ⑥ 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 Molecular Biology

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) * 2/27/2021 Molecular Biology 5

Fundamentals of Gene Expression ① 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 Molecular Biology

② Processing also presents a problem for bacterial expression of human m. RNAs. The eukaryotic ORF must be cloned into the expression vector, not the genomic copy ② The genetic code is identical for most bacterial and eukaryotic m. RNAs. Although the code is the same, expression levels can be affected by codon frequency, which varies between organisms and transcripts ④ 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) Molecular Biology

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 3 D “Native” conformation Lack PTMs (Post translational modifications) needed for specific activity Molecular Biology

Which Vector? Promoters * arabinose systems (p. BAD), phage T 7 (p. ET), Trc/Tac promoters, λ PL or PR Tags标签 * * * * His 6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coil/K-coil tags (poly E 35 or poly K 35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags Molecular Biology

Fusion Protein Gene T 7 Promoter RBS ( with / without stop codon ) ATG TAG lac Operator peptide tag: V 5 / poly-(his) mutiple cloning site (mcs) peptide tag: V 5 / poly-(his) Molecular Biology

A Generic Vector Molecular Biology

II. Eukaryotic Expression System 1. Yeast expression 2. Insect expression system 3. Mammalian expression system Molecular Biology

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 Molecular Biology

Yeast Expression Vector Comments for p. PIC 9 K: 9276 nucleotides 5´ AOX 1 promoter fragment: bases 1 -948 5´ AOX 1 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´ AOX 1 primer site: bases 1327 -1347 3´ AOX 1 transcription termination (TT): bases 1253 -1586 HIS 4 ORF: bases 4514 -1980 Kanamycin resistance gene: bases 5743 -4928 3´ AOX 1 fragment: bases 6122 -6879 p. BR 322 origin: bases 7961 -7288 Ampicillin resistance gene: bases 8966 -8106 Molecular Biology 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) * Molecular Biology

Cloning in Yeast Cells Uses a special plasmid that works both in E. coli and Yeast ② Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isn’t circular) ③ 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 ④ Now gene of interest is under control of the powerful AOX promoter ① Molecular Biology

Cloning 2 1 3 4 2/27/2021 Molecular Biology 17

2. Insect Expression Pros 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 Molecular Biology

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 * Molecular Biology

Baculovirus (Ac. MNPV) Cloning Process Transfer vector Cloned gene 5’ 3’ x x Cloned gene 5’ 3’ Polyhedrin gene Ac. MNPV DNA 2/27/2021 Molecular Biology Recombinant Ac. MNPV DNA 20

Baculovirus Molecular Biology

Bac. Virus Expression System p. IEx™Insect Cell Expression Plasmids (Novagen) * Bac-NBlue™Baculovirus Expression System (Invitrogen) * 2/27/2021 Molecular Biology 22

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 * * * Molecular Biology

He. La cells in culture 2/27/2021 Molecular Biology 24

He. La cells from the Nikon microscope web site 2/27/2021 He. La 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 1950 s. 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 Molecular Biology 25 radiation.

He. La Human cells JONATHON PINES REGULATION OF MITOSIS IN MAMMALIAN CELLS Mitotic He. La cell stained with anti-Cks 1 (red), anti-tubulin (yellow) and DAPI (blue) 2/27/2021 Molecular Biology 26

He. La cells as you will see them Expression of (A) b-galactosidase and B green fluorescent protein in He. La cells. Cells were transfected in 6 -well plates. Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection. 2/27/2021 Molecular Biology 27

Expression System Selection Choice depends on size and character of protein * * * Large proteins (>100 k. D)? Choose eukaryote Small proteins (<30 k. D)? 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 Molecular Biology

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 Molecular Biology

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 * Molecular Biology

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 * Molecular Biology

Methotrexate (MTX) Selection Gene of interest DHFR Transfect dfhr- cells Grow in Nucleoside Free medium 2/27/2021 Culture a Colony of cells Molecular Biology Grow in 0. 05 u. M Mtx Culture a Colony of cells 32

Methotrexate (MTX) Selection Grow in 0. 25 u. M Mtx 2/27/2021 Culture a Colony of cells Grow in 5. 0 u. M Mtx Molecular Biology Culture a Colony of cells Foreign gene expressed in high level in CHO cells 33

Mammalian Expression Systems Comments for pc. DNA™ 4/TO : 5078 nucleotides CMV promoter: bases 232 -958 TATA box: bases 804 -810 Tetracycline operator (2 X Tet. O 2) 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 f 1 origin: bases 1365 -1793 SV 40 promoter and origin: bases 1803 -2143 EM-7 promoter: bases 2183 -2249 Zeocin™ resistance gene: bases 2250 -2624 SV 40 early polyadenylation sequence: bases 27542884 p. UC origin: bases 3267 -3937 bla promoter: bases 4937 -5041 (complementary strand) Molecular Biology 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 * Molecular Biology

Summary 1. 2. 3. 4. 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 Molecular Biology