of the receptor-associated protein (RAP) D3 domain. J. Biol. Chem. 290:17262-
Punt PJ, van Biezen N, Conesa A, Albers A, Mangnus J, van den Hondel C. 2002. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol. 20:200–206.
Qiu J, Swartz JR, Georgiou G. 1998. Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl. Environ. Microbiol. 64:4891–4896.
Robinson AS, Hines V, Wittrup KD. 1994. Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae. Bio/Technology. 12:381–384.
Rogers S, Wells R, Rechsteiner M. 1986. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 234:364–368.
Rosano GL, Ceccarelli EA. 2014. Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol. 5:1–17.
Schröder M. 2008. Engineering eukaryotic protein factories. Biotechnol. Lett. 30:187–196.
Seo JH, Bailey JE. 1985. Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol. Bioeng. 27:1668–1674.
Shi X, Jarvis DL. 2007. Protein N-glycosylation in the baculovirus-insect cell system. Curr. Drug Targets. 8:1116–1125.
Simmons LC, Yansura DG. 1996. Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli. Nat. Biotechnol. 14:629–634.
Steinborn G, Gellissen G, Kunze G. 2007. A novel vector element providing multicopy vector integration in Arxula adeninivorans. FEMS Yeast Res. 7:1197–1205.
Strausberg SL, Alexander PA, Gallagher DT, Gilliland GL, Barnett BL, Bryan PN. 1995. Directed evolution of a subtilisin with calcium-independent stability. Bio/Technology. 13: 669–673.
Tobias JW, Schrader TE, Rocap G, Varshavsky A. 1991. The N-end rule in bacteria. Science. 254:1374–1377.
Van Oers MM, Pijlman GP, Vlak JM. 2015. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology. J. Gen. Virol. 96:6–23.
Wang L, Brock A, Herberich B, Schultz PG. 2001. Expanding the genetic code of Escherichia coli. Science. 292:498–500.
Wilkinson DL, Harrison RG. 1991. Predicting the solubility of recombinant proteins in Escherichia coli. Bio/Technology. 9:443–448.
Wu X-C, Ye R, Duan Y, Wong S-L. 1998. Engineering of plasmin-resistant forms of streptokinase and their production in Bacillus subtilis: streptokinase with longer functional half-life. Appl. Environ. Microbiol. 64:824–829.
Zhang G, Brokx S, Weiner JH. 2006. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nat. Biotechnol. 24:100–104.
review questions
1. What DNA sequence elements are required for expression of a cloned gene in a prokaryotic host?
2. What is a strong promoter? Why is a strong promoter not always desirable for expression of a cloned gene?
3. What is a regulatable promoter? How is the E. coli lac promoter used to regulate the expression of a clone gene?
4. The promoter for gene 10 of the E. coli bacteriophage T7 is an example of a strong promoter. How is it used to express a cloned gene?
5. Why is codon optimization often required for production of high levels of a recombinant protein?
6. What are inclusion bodies, and how can their formation be avoided?
7. How can a protein of interest be engineered to be secreted to the medium by E. coli?
8. Discuss some strategies to purify a recombinant protein produced in a prokaryotic host. Consider that a protein may be used as a human therapeutic agent.
9. Why is it sometimes advantageous to integrate a target gene into the chromosomal DNA of a prokaryotic host? How might this be achieved?
10. During the course of integrating a target gene into the chromosomal DNA of the host bacterium, a marker gene may also be inserted into the chromosomal DNA. What strategy could be used to excise only the marker gene?
11. What are the major posttranslational modifications of eukaryotic proteins in the endoplasmic reticulum and Golgi apparatus?
12. Describe the features of a eukaryotic expression vector.
13. What criteria are used to decide if a particular recombinant protein should be produced in a yeast, insect, or mammalian cell system?
14. What are the advantages and disadvantages of the different classes of yeast vectors for producing a biotechnology product?
15. Describe some of the strategies that have been used to increase proper folding and secretion of recombinant proteins from yeast cells.
16. Discuss the salient features of a P. pastoris high-expression integrating vector system. How has P. pastoris been “humanized”?
17. What are baculoviruses?
18. Describe a strategy that can be used to insert a target gene into the baculovirus genome for expression in insect cells.
19. Describe the main features of an extrachromosomal mammalian-cell expression vector.
20. Why are yields of recombinant proteins produced by mammalian cells in large bioreactors generally low? How can yields be improved?
21. What is chromatin, and how does it affect gene expression? Describe some of the strategies that have been developed to increase expression levels of a target gene that is integrated into a chromosome of a eukaryotic host cell.
22. You have produced a recombinant enzyme for an industrial application but have found that the protein is unstable at moderately high temperatures. Describe how you would increase the stability of the enzyme. Assume that you have determined the DNA sequence of the gene encoding the enzyme and the structure of the enzyme.
23. How can unusual amino acids be incorporated into proteins, thereby producing an altered form of the target protein?
24. You have produced a recombinant enzyme for an industrial application but have found that the catalytic activity is low. Describe how you would increase the activity of the enzyme if the DNA sequence of the gene was known but the structure of the enzyme was not determined.
25. Outline two ways in which DNA shuffling may be used to generate hybrid genes.
26. How would you engineer streptokinase so that it was less sensitive to proteolytic digestion?
27. How can the gene(s) encoding a Fab fragment of a monoclonal antibody be modified so that the specificity of the antibody is altered?
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