Engineering Of E. Coli For Synthesis Of Mannose-6-Phosphate And Spider Silk Protein MaSp2

Mannose-6-phosphate is an important phosphorsugar,which is involved in many physiological functions and it is used to treat many diseases.Its production is however expensive since it requires ATP a costly substrate as phosphorylation agent.This study has focused upon the direct synthesis of M6P by enzyme glucomannokinase using inorganic polyphosphate without involvement of ATP.The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression.After purification involving affinity chromatography,a thick band of 30kDa related to the enzyme has been purified from alloyed supernatant.A total amount of 0.69 mg/ml of enzyme has been successively obtained and the purity exceeds 90%.The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose,as it can be visually seen from the KM values 1.7,9.5,4.6 and 203.7 ?M,respectively.The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5,temperature 25?,poly(P)/mannose ratio 3:1 and in the presence of bivalent ion Mg2+.This study supported the evidence that the glucomannokinase from Mycobacterium phlei is suitable for further production of mannose-6-phosphate.Additionally,another research was also carried out in this project in which MaSp2 of spider silk protein was produced As silk is an important biological structure,which can used to make many important biomaterials such as sutures and has two important protein,major ampullate spidroin 1 and 2(MaSp1&2).This study is performed to produce MaSp2 artificially,(taking advantage of advance metabolic engineering techniques)using Escherichia coli BL21(DE3)as host.Initially a monomeric unit of silk gene(up to 100 bp)called gene unit(GU)was constructed.Subsequently,this short single-stranded oligonucleotide GU was subjected to head to tail ligation in order to synthesized GU of different length from 2-64 and was inserted in to plasmids.Two plasmids named pET28a(+)and pET22b(+)contained MaSp2 and gene of Ala/glyc-tRNA respectively,were co-expressed in Escherichia coli BL21(DE3),that grown in a medium having 0.05%of alanine and glycine(to meet extra demand,so early stoppage of translation could avoided),resulting artificial MaSp2 of 55 and 110 kDa was achieved.This study exhibited a successful example of the heterologous system that has attained the expression ofthe protein which has of higher molecular weight and the repeated amino acids motifs in Escherichia coli.