Investigation of recombinant Lactate Dehydrogenase Expression using SUMO fusion tag

Background and Aim: Lactate dehydrogenase (LDH; NAD oxidoreductase, E.C ۱.۱.۱.۲۸), an important enzyme which acts in the final step of glycolysis, has been expressed as recombinant enzyme due to its importance in industry by different researchers since ۱۹۸۰s. However, difficulties in expressing soluble proteins is a complex and critical issue. Fusion systems (e.g. ubiquitin) have been designed and been introduced to increase the solubility of their target protein. SUMO (Small Ubiquitin-like Modifier) tag has been developed as an alternative fusion partner which could circumvent the obstructions of ubiquitin tags (e.g. high cost and obtainability of ubiquitin-specific proteases). Here, in order to investigate the solubilization effect of SUMO tag on expression of recombinant LDH enzyme we fused the gene sequence of SUMO-tag to the LDH sequence.Methods: In order to obtain LDH gene, PCR technique was performed using proper primers which added Bam HI and Xho I restriction sites as SUMO sequence was available inside pET-۲۱ between Nde I and Bam HI restriction sites. The amplicon and SUMO construct then were subjected for double digestion using Bam HI and Xho I. Digested products then were subjected for ligation using T۴ DNA ligase, and then was transformed into competent E. coli BL۲۱ using heat-shock method. To evaluate and determine the best expression condition for SUMO-LDH and LDH, both proteins were expressed in LB and TB medium in different expression condition such as ۴ h, ۸ h, and ۱۸ h of incubation at ۱۴°C, ۲۳°C, and ۳۷°C after inducing the expression by ۰.۱ mM of Isopropyl β-D-۱-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the expression difference between LDH and SUMO-LDH proteins.Results: Succeeding PCR which resulted in the generation of LDH containing Bam HI and Xho I restriction sites, double digestion was performed successfully for SUMO construct and amplicon by Nde I, Bam HI and Bam HI, Xho I, respectively. Following expressing the enzymes in different conditions, SDS-PAGE illustrated a difference between expression of LDH and SUMO-LDH which will be discussed.Conclusion: As SUMO-tag may enhance the expression and solubility of LDH, the mentioned method can be used for LDH production in diagnostic kits.

کلیدواژه ها:

Lactate dehydrogenase ، Small Ubiquitin-Related Modifier Proteins ، Escherichia coli ، Solubility