Influence of Specific Signal Peptide Mutations on the Expression and Secretion of the α-Amylase Inhibitor Tendamistat in

Abstract

The Streptomyces ␣-amylase inhibitor tendamistat is secreted by a signal peptide with an amino-terminal charge of ؉3. To elucidate the influence of the charged residues on protein secretion in Streptomyces, the amino-terminal charge was varied from ؉6 to neutral net charge. The effects of charge variation were analyzed in combination with three Streptomyces promoters and two transcriptional terminators. Introduction of additional positive charges significantly decreased the amount of secreted tendamistat. On the contrary, a charge reduction to ؉2 resulted in the doubling of inhibitor production. After exclusion of transcriptional effects, the observed alterations of inhibitor secretion by the mutants with a charge of ؉6 to ؉2 were attributed to a modulation of precursor synthesis. Furthermore, a tight coupling of synthesis and export was stated. Charge reduction to ؉1 or neutral charge generally reduced the yield of secreted tendamistat, yet remarkable differences were found for mutants with identical net charge. Elimination of the positive charge at a defined position resulted in the release of tendamistat precursor protein, which suggested a specific uncoupling of synthesis and translocation.

Signal peptides for recombinant protein secretion in bacterial expression systems

“…In line with this notion, the yields of mouse tumor necrosis factor secreted by S. lividans could be increased sevenfold by increasing the net charge of the n-region of the signal peptide from a Streptomyces venezuelae α-amylase from + 2 to + 3 [ 68 ]. In contrast, a decrease of the positive net charge of the n-region of the Streptomyces tendae α-amylase inhibitor tendamistat signal peptide from + 3 to + 2 resulted in the doubling of the amounts of tendamistat in the supernatant of the heterologous expression host S. lividans , whereas increasing the positive net charge to + 4 or + 6 had an adverse effect [ 69 ]. Similarly, a saturation mutagenesis of the positions 2–7 of the n-region of the B. subtilis α-amylase AmyE signal peptide revealed that three out of four isolated mutant signal peptides that significantly increased the amounts of the heterologous target protein cutinase from F. solani pisi in the B. subtilis culture supernatant likewise led to a reduction of the net charge of the n-region from + 3 to + 2 [ 70 ].…”

Expression byStreptomyces lividansof the RatαIntegrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

“…As described by Mhiri et al [10], the long synthetic signal peptide (LSSP) contains two positive charges on the N-terminal region and carries two ribosome binding sites (RBS) as well as two translational initiation codons which are known to contribute to increasing the yield of the secreted protein [15, 32]. This signal peptide was successfully used for the secretion of the Streptomyces sp.…”

Inheritance stability of hybrid plasmid TrS16 in transformant under long-term culture conditions without supporting selection

“…Существует 2 типа нестабильности плазмид in vivo: сегрегационная -утрата в процессе деления клеток, и структурная -изменение структуры. Также установлена важная проблема шаттл векторов -нестабильность наследования в клетках стрептомицетов [1, [7][8][9]. Однако устойчивость к тиострептону клеток трансформантов может обеспечиваться присутствием в них как гибридных плазмид, так и вектора.…”

“…Проведен анализ молекулярного размера автономных плазмидных ДНК 12 Tsr R колоний, выбранных произвольно. Было установлено, что плазмиды 9 изученных клонов имели молекулярный размер, соответствующий размеру HindIII/EcoRI фрагмента плазмиды pAX5a [7]. Плазмиды же 3 клонов сохранили размер (8,4 т. п.…”

“…Даная рекомбинантная плазмида создана с использованием плазмиды pАХ5а, которая содержит в своей конструкции фрагмент (essential origin) стрептомицетной плазмиды pIJ101, кодирующий информацию, необходимую для репликации и наследования плазмиды [2,3,7,9]. Установлено, что векторы, содержащих аналогичный фрагмент плазмиды pIJ101, характеризуются стабильностью наследования (pIJ702, pIJ486, pIJ211, pIJ303 и множество других).…”