Chapter 1 Post-translational modifications by S. cerevisiae ‘The heterologous proteins synthesized by S. cerevisiae undergo. postiransational changes ‘while they are being exported into the exvacellular environment. To faciliate protein. secretion, 2 Single (leader) peptide is attached 10 the protein, This peptide is removed by the yeast endoprotesse. OTHER YEAST EXPRESSION SYSTEMS Despite the-very succesful use ofS, cerevisiae for generating recombinant proteins, there are Certain limitations. These include a very low or a limited yield, dificult in seeetion of some proteins and hyperslycosyation. Attempts are being made ta explore the utility of other yeasts for the production of hepatitis B virus surface antigen [H8sAg) and bovine Ieozyme, The yeast Hansenula ha, is employed. for the synthesis of o and Beglobin chains of human hemoglobin. * =a INSECT CELL EXPRESSION SYSTEMS. CGltured insect cells are in use for expressing cloned DNAS. Baculovinuses exclusively infect insect cells. The DNA of these viruses encode for several products and their productivity. in cells is very high to the extent of more than 10,000 times compared to mammalian cells. Besides carrying large number of foreign genes, the baculoviruses can effecvely express and process the products formed. Another advantage with these viruses is that they cannot infect humans, oes vertebrates or plans. This, Baculoviuses are safe vectors. Polyhedein gone of baculovirus The polyhedsin gene is responsible for the synthesis of a matrix protein-polyhedrin. This protein is synthesized in large quantities by baculovirus during the infection cycle. Polyhedrin protects the virus ‘fom being inactivated by environmental agents. The promoter for polyhedrin gene is very strong. However, the lifecycle of baculovirs does not depend on the presence of this gene. Polyhedrin gene can be replaced by @ Cloned gene, and the genetically engineered baculovirus can infect the cultured insect cells. The MANIPULATION OF GENE EXPRESSION IN. HOST CELLS sat Tobe 1 Seeded cane of cman ‘proteins produced by baculovirus expression ‘vector system ‘eanosne daninace [Akane phosphatase ‘Anyi preouser rela ‘Aneasanigan DNA po}ymarase Enyhropoitn HIV emeloe pata Interns (0, B) Irereukn:2 Malai poains Pancreat pase Polo vius prin Rabies vrs proteins Radon San rtaius capsid arigen sue plasminogen activator Cloned. gene expresses, and large quantities of recombinant proteins ate praluced, Because Of close similarity inthe posetransltional modifications between insects and mammals, biologically active proteins can be produced by this approach, And in fact, by using baculovirus 3s fan expression vector system, a good number of mammalian and vial proteins have been synthesized (Table 11.0, Baculovirus expression vector system ‘The most commonly used baculovius. is Autographa californica multiple nuclear polyhedrosis virus (ACMNPY). i can grow on the Insect ell lines (eg, derived from fall army worm) land produce high’ levels of polyhedrin or a recombinant protein, ‘The organization of a baculovirus (ACMNPY) ‘eansfer vector is showe in Fig. 11.34. It consists of an E. colibased plasmid vector alongwith the DNA of baculovitus. This in turn has AeMNPV DNA, a polyhedrin promoter region, cloning site far inert ONA and polyhedrin termination region, ‘When the insect culture cells, transfected with |AcMINPV are mined with transfer vector carying 2 cloned gene, a double crossover occurs. The result is that the cloned gene with polyhedrin promoter and temination sequences gets integrated into142 “ VectorONA SARIN Fp Sater. a BIOTECHNOLOGY Ce Pt S-AQMNEY Vector DNA BNR Tanger ee neti ene es ‘ene Sopp mes mY “oe one 1 Prine Fig. 113 : Aeculovius xaveeson voce” system (A) Organlzaton of bacuovins rarer vector (B) Replacement ‘of he poyesn gene of bacuouts with a caned gano fom a arse vector (AcMNPV-Auiographa callemiza ‘nulile nidesr poyhedrosie ru; Pp-Palpyhn gene prams; Os-Ciong sit: P-Payhedn gene teminaon: Nate: The cading region of polechn gene not shown in A). ACMNPV DNA (Fig. 1138. In this process, polyhedrin ene is lost. the recombinant Fbaculovitus containing cloned gene is isolated. The host insect culture cells, on infection with recombinant baculovirus, produce heterologous Broteins. A lage number and a wide varity of recombinant proteins (around 500) have been symbheszed in the laboratory A majority of them 95%) have the requisite postranslational modifications. A seleced. it of recombinant proteins is given in Table 11.1 Modifications in the production of recombinant baculovirus The original method of creating recombinant baculovirus has undergone several changes. Incorporation of a unkque su 361 restriction ‘endonuclease se onthe polyhedrin gene increases the vied of recombinant baculavius production to about 30% from the normal 1%. Bacmid : This is shutle vector for E. coll and insect cell baculovirus. Construction of a recombinant bacmid Is a novel approach to carry MiChapter 11 MANIPULATION OF GENE EXPRESSION IN HOST CELLS. 4143 Chapter 11_: MANIPULATION OF GENE EXPRESSION IN HOST CELLS _443 = pe one Pes 7a 114 7A dag eprsonaton of mannan espace voor POM 0:08 ‘ru pojadnyaton sequen re=-tiecem wis en Savenne art’ gon, OrP-0700 of ‘eukarynicraptcation: OrfOngin of ool aplication: mc inte gna ‘out all the genetic manipulations including the expression of baculosirus vector in Ecol Use of yeast cells: The genetic manipslations of ‘AcMNPV genome can be done in yeas cells with Yyeastinect shutle vector, Then the recombinant haculovirs i intraduced into insect cel MAMMALIAN CELL EXPRESSION VECTORS, Mammalian expression vectors are useful for the production of specific and authentic recombinant proteins (for use as therapeutic ‘agents. In. addtion, they are also. helpful for Studying the function and regulation of mammalian genes. In. general, the mammalian. expression Vectors are quite comparable to other eukaryotic fexpresion vectors. However, _ large-scale production of recombinant proteins with fenginered marsmalian cals is costly 'A diagrammatic representation of mammalian vector is shown in Fig. 11.4 Iecontains a eukaryotic {Fin of repletion nn a animal vias auch 2 Simian virus 40 ($V40) and a prokaryotic origin of, replication (om €, col), The mammalian vector has a multiple cloning ste and a selectable marker gene. Both of them are under the control of eukaryotic promoter and polyadenylation Sequences, These sequences are obtained from tither animal viruses (SVAD, herpes simplex virus for mammalian genes (growth hormone, metallothionein). The promoter sequences facilitate the transcription of cloned genes (at the motile cloning sie) and the selectable marker genes. On the other hand, the polyadenylation sequences terminate the transcription. Ampicilin resistant marker gene can be used for selecting the transformed E.coli cells. Markers for mammalian expression vectors “There are several markers in use forthe selection of tanstred mammalian cells The bacerll yen {Neo that encades for neomycin phosphotrans- ferase is frequently used. The other markers ae the tenes that encode for the enzyme dihydrofolate Feductase (OHFR), and glutamine synthetase (CS) Several. proteins have been produced by snammalian expression vectors, The yield of protein Synthesis In some cases, i increased by Inserting an intron between the promoter and the cloned gene. However, the reason for this fs not clear. Coordinated expression By coordinating the expression of a cloned gene and” a. selected marker, the production of recombinant protein can be increase. ‘The coordinated expression of DHFR marked) and a cloned gene is ilestrated in Fig. 11.5. The DHFR gene i inserted close toa cloned gene and both of them are under the control of a single promoter and termination (polyadenylation) fequences. The DHFR gene is flanked by inton Femoving sequence. As the genes are expressed, DHER is synthesized by primary transeript while the recombinant protein is formed from spliced mRNA. Expression of two cloned genes Some protsins are composed of two or more subunits. For example, hemoglobin is 2 tetramer With two copies of each subunit Le, cy. Each fubunit can be separately synthesized by the Corresponding cloned gene, and they are then Irixed to form the mlimeric protein in vitro. This. ‘method is not very satisfactory since the In vitro sembly” of multimeric proteas not efficent ‘Techniques have been developed in recent years 0 produce two diferent proteins (he, subunits of 3 frultimeric protein) in the same cel simultaneously.