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Ribonuclease A recombinant expression and refolding studies

Author: XiaoWenChu
Tutor: DongXiaoYan
School: Tianjin University
Course: Biochemical Engineering
Keywords: Ribonuclease A Expression Metal chelate chromatography Histidine tag Refolding
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 45
Quote: 2
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Abstract


Ribonuclease A (Ribonuclease A, EC3.1.27.5, RNase A) Preparation of DNA, inhibition of viral replication and the model as a basis for protein has a wide range of uses and needs, but the commercialization of RNase A are isolated from the pancreas of animals obtained was purified, which not only limits its access to a large number, but also result in complex processes and high production costs. Therefore, the use of genetic engineering to obtain a large number of biologically active RNase A has become a research hotspot. Reported in the literature in the expression of RNase A multi-soluble form through the expressed less, which limits their industrial production and application. Based on this, this study PCR from genomic bovine pancreatic RNase A was amplified coding sequence, insert it into efficient with histidine-tagged expression plasmid pET28a, and then using the E.coli BL21 (DE3) to highly expressed in the form of inclusion bodies, after dilution and the use of metal chelate chromatography (IMAC) for the renaturation of inclusion bodies, provide a theoretical basis for industrial applications and experimental basis. The results show that using PCR techniques from the genome of bovine pancreatic RNase A gene was amplified and sequenced with tissue DNA showed a homology of 100%; this target gene is inserted into the vector pET28a, transferred E.coli BL21 (DE3) host bacteria obtain pET28a-RNase A genetically engineered to express bacteria; RNase A gene inserted into genetically engineered bacteria without affecting growth pattern, after induction, finding the target protein mainly expressed in the form of inclusion bodies; goals Protein expression of the optimal conditions: bacterial culture OD600 of 0.4 (about culture 2 h, cells in the logarithmic growth at this time pre) started induced expression, IPTG concentration of 1 mM, induced 3 h, this condition RNase A expression of the inclusion body was 60 mg / L culture solution; containing body through urea, surfactant and the wash buffer PE, the ultimate goal of protein purity of 83%. The presence of RNase A contains the optimal denaturation time (1 h); refolding allows only low concentrations of protein (≤ 0.1 mg / mL) to obtain better refolding (renaturation 24 h, activity yield was 52 %); IMAC method allows a final concentration of protein 0.5 mg / mL of RNase A refolding after 24 h to reach 80% of the activity recovery, while achieving the RNase A inclusion body purification (refolded product purity of 97%) . And refolding compared to the same final protein concentration (0.5 mg / mL) under, IMAC France refolding yield than refolding increased by nearly 60 percentage points, the purity of the product is also increased by 14 percentage points. In addition, by circular dichroism spectroscopy performed on the refolding product analysis showed. Refolded RNase A and RNase A natural high-level structure similar.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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