Comparison of the expression of native and mutant bovine annexin IV in Escherichia coli using four different expression systems.
Bovine annexin IV, a Ca(2+)-dependent, membrane-binding protein, was expressed in E. coli using four different prokaryotic expression vector systems. An annexin IV cDNA was mutated in the 5' noncoding region to introduce an NcoI restriction site at the translation initiation site. The coding sequence was then excised and ligated into the expression vectors: pKK233-2 (which uses a hybrid trc promoter), pFOG405 (which uses the alkaline phosphatase promoter and generates a fusion protein with the alkaline phosphatase signal sequence that targets the protein for secretion), pOTSNco12 (which provides temperature-sensitive expression from the lambda phage promoter), and pET11d (which uses the T7lac promoter and a protease-deficient host). Expression of wild type and mutant annexin IV in the various systems was compared. Differences in level of expression, formation of inclusion bodies, and yield of purified protein were observed. The pET11d system was found to be the most effective expression system for annexin IV and various annexin IV mutant constructs, providing the highest yield of functional protein from the soluble fraction of cell lysates. Bovine chromaffin granule binding and aggregating activities of recombinant annexin IV were found to be virtually indistinguishable from those of bovine annexin IV isolated from liver tissue. Truncation constructs containing one, two, or three of the four conserved 70-amino-acid domains of native annexin IV were successfully created and expressed in E. coli, but the recombinant proteins were generally insoluble. pET11d annexin constructs containing point mutations in residues involved in binding calcium produced soluble protein at levels comparable to those of constructs expressing wild type protein.
Nelson
M R
MR
Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
Creutz
C E
CE
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017314:Annexin A4 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics*; D002479:Inclusion Bodies; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014157:Transcription Factors / Q000235:genetics; D014764:Viral Proteins
D017314:Annexin A4; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C039000:cII protein, bacteriophage lambda; D007544:Isopropyl Thiogalactoside; D000469:Alkaline Phosphatase