ABSTRACT
In this review, we summarize some of our results on folding and directed evolution of an antibody fragment in Escherichia coli cytoplasm. We will also discuss some attempts to construct other antibodies active in this cellular compartment.
Subject(s)
Cytoplasm/metabolism , Directed Molecular Evolution , Escherichia coli/genetics , Immunoglobulin Fragments/metabolism , Protein Folding , Escherichia coli/metabolism , Immunoglobulin Fragments/geneticsABSTRACT
The pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1) has been involved in the survival and differentiation of neuroblasts during development. This study examined the effects of various neurotrophins on the activity of the mouse PAC1 promoter/luciferase reporter constructs in rat PC12 cells and in 8-day-old mouse cerebellar granule cells. In PC12 cells, both differentiating factors such as nerve growth factor (NGF) and mitogens such as epidermal growth factor (EGF) and insulin growth factor-1 (IGF-1) up-regulated PAC1 promoter activity by 2-4-fold in a concentration-dependent manner. Although PACAP differentiated the PC12 cells, it had no effect on the PAC1 promoter and antagonized the stimulatory effect of NGF. In cerebellar granule cells, IGF-1 and brain-derived neurotrophic factor (BDNF) also stimulated the activity of the PAC1 promoter. NGF and IGF-1 increased endogenous PAC1 mRNA levels, and the NGF-induced up-regulation is the result of an increase in transcription from PAC1 promoter instead of an increase in mRNA stability. The mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, prevented the transcriptional effects both in PC12 and cerebellar granule cells. Moreover, expression of dominant-negative Ras protein in PC12 cells also prevented the NGF effect. Our results show that the PAC1 promoter can be up-regulated by diverse neurotrophins via an MAPK-dependent pathway and suggest a role for the Ras protein.
