Transcription analysis and cloning of u32.002 collagenolytic peptidase from geobacillus lituanicus dsm 15325t

Abstract

Collagens are one of the most important proteins including their functions and biotechnological application. The biotechnological application of collagens wouldn‘t be possible without the process of collagenolysis which is catalysed by collagenolytic peptidases. The aim of this work was to perform transcriptional analysis of U32.002 (Helicobacter-type) collagenolytic peptidase gene and its cloning as well as basic analysis. The analysis of U32.002 peptidase gene transcription was performed after the extraction of total RNA from G. lituanicus DSM 15325T cells that were in two different stages of growth. Reverse transcription assays were performed after total RNA extraction. After the process of reverse transcription samples of cDNA were used for the diagnostic PGR that was carried out by using five different primers. This gene was cloned with and without putative signal/pro sequence in order to produce the preparation of U32.002 peptidase. Full sequence of U32.002 peptidase gene was cloned into pTZ57R/T and then into the expression vector pET28c(+). U32.002 gene without putative signal/pro sequence was cloned into pJET1.2, and then into pET28c(+). SDS-PAGE and MALDI-TOF analyses were performed in order to determine the fact of expression in E. coli BL21 (DE3). Full scale expression optimisation was performed, as well. The influence of calcium and zinc ions to the structural stability of U32.002 peptidase with putative signal/pro sequence was analysed. The collagenolytic activity of U32.002 peptidase was analysed by using zymography of native collagen, type I. The U32.002 peptidase of G. lituanicus DSM 15325T is transcribed constitutively during the exponential phase of growth, whereas only fragments of U32.002 gene transcript are observed in the later growth phases. It is evident that zinc ions increase structural thermostability of U32.002. The homodimer of U32.002 peptidase with putative signal/pro sequence was able to digest native type I collagen at 50 °C; pH 7,4