Abstract
High relative mutability may be a common property of the surfaces of all or most proteins and may be exploited during evolution not only to alter molecular recognition but to modify catalytic functions as well. Conservative amino acid substitutions often can be expected to cause minimal structural alterations, but the properties of protein surfaces and the mechanisms of protein folding that accommodate length variation without loss of function are not understood. To begin to study these aspects of protein structure and folding, we have constructed short amino acid insertions in the Escherichia coli alkaline phosphatase polypeptide by linker insertion mutagenesis of the phoA gene and have examined correlations between mutant protein function and position of the insertions relative to the x-ray map of wild type alkaline phosphatase determined by Wycoff and colleagues (Sowadski, J. M., Foster, B. A., and Wycoff, H. W. (1981) J. Mol. Biol. 150, 245-272). Mutant protein enzymatic function was generally tolerant of insertions in exterior loops, but was inactivated by insertion within alpha-helical or beta-strand structural elements. We further demonstrate that these tolerant surface loops can serve as vehicles for high level expression and stabilization of larger foreign peptide sequences, using a 15-residue analogue of dynorphin as an example. Insertion of the dynorphin "guest" peptide probably caused only a local structural perturbation of the alkaline phosphatase carrier since the hybrid protein retained enzymatic activity, was exported efficiently to the periplasmic space, and could be purified by anion-exchange chromatography using a protocol developed for alkaline phosphatase itself. The gust peptide was recovered from one of these fusion proteins intact and in high yield by protease digestion in vitro and was then purified by cation-exchange chromatography to near homogeneity in a single step.
Highlights
Introduction of Guest Peptides intoEscherichia coli Alkaline PhosphataseEXCISION AND PURIFICATION OF A DYNORPHIN ANALOGUE FROM AN ACTIVE CHIMERIC PROTEIN*(Receivedfor publication, July 21,1989)Paul I
The mutations were characterized by restriction enzyme mapping and local DNA sequencing, and their effects on alkaline phosphatase (AP) function were assessed by expression of mutant genes in E. coli strain AW1043, which carries a deletion in the chromosomal phoA gene [10]
In this paper we have demonstrated that insertion of di- or tripeptides within the E. coli AP primary structure often does not decrease the activity of the enzyme significantly, if the insertions are directed to sites on the molecular surface of the wild type tertiary structure
Summary
Introduction of Guest Peptides intoEscherichia coli Alkaline PhosphataseEXCISION AND PURIFICATION OF A DYNORPHIN ANALOGUE FROM AN ACTIVE CHIMERIC PROTEIN*(Receivedfor publication, July 21,1989)Paul I. High relative mutability may be a common property of the surfaces of all or most proteins and may be exploited during evolution to alter molecular recognition but to modify catalytic functions as well. Conservative amino acid substitutions often can be expected to cause minimal structural alterations, but the properties of protein surfaces and the mechanisms of protein folding that accommodate length variation without loss of function are not understood. To begin to study these aspects of protein structure and folding, we have constructed short amino acid insertions in the Escherichiu coli alkaline phosphatase polypeptide by linker insertion mutagenesis of thephoA gene and have examined correlations between mutant protein function and position of the insertions relative to the x-ray map of wild type alkaline phosphatase determined by Wycoff and colleagues
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