chapter  2
55 Pages

Use of Recombinant DNA Technology for Engineering Mammalian Cells to Produce Proteins

ByRandal J. Kaufman

This chapter focuses on the use of recombinant DNA technology in the engineering of mammalian cells to express high levels of proteins from heterologous genes. It addresses the various methods for transfer of genetic material into mammalian cells and methods to select for stable transformants followed by selection for gene amplification. The chapter describes important components in the design of expression vectors, such as potential for gene amplification, transcriptional controls, mRNA processing, and translation signals. Potential limitations which result from the requirement for specific posttranslational modifications and the influence of the host cell in obtaining appropriate expression and secretion are discussed. Two general pathways of secretion exist in higher cells: the constitutive pathway and the regulated pathway. All cells carry out constitutive secretion and secrete proteins with a variety of posttranslational modifications. A variety of posttranslational modifications can occur in the Golgi apparatus.