Expression, Purification And Preliminary Bioactivity Of Fusion Protein GGH Analogues With N-terminal Extension

GGH is a fusion protein of two human glucagon-like peptide-1mutant and human serumalbumin. Compared with GLP-1, it can not only stimulate the searetion of insulin and thegrowth of β cells but also show longer half-life than GLP-1. However, it did not effect asexpected because of its incomplete N-terminal residues. In this study we designed five GGHanalogues with N-terminal extension and constructed five recombinant plasmidspPIC9K/GGH-analogues capable of expressing the analogues in the methylotrophic yeast P.pastoris GS115. GGH analogues were purified and screened through activity test in vitro andin vivo. The conclusions are mainly as follows:(1) Based on the protein GGH, five analogues with N-terminal prolongation weredesigned. Primes for PCR were firstly designed and synthesized and the genes of GGHanalogues were obtained by polymerase chain reaction. Then the recombinant expressionplasmids were constructed after broth PCR、double digestion conformation as well as DNAsequencing. Finally the recombinant strains P.pastoris GS115capable of secreting GGHanalogues were screened on MD plate.(2) The recombinant P.pastoris GS115were cultured in conditions as follows:120mL/flask、 medium pH6.0、30℃、200r/m、2%methanol、induced for72h. The expressionquality of the five recombinant strains reached150to160mg/L in flask-scale fermentation.(3) The fusion proteins were successfully purified from the supernatant of the brothusing methods combining ultrafiltration concentration, affinity absorption chromatography,hydrophobic chromatography, ion exchange chromatography and gel filtration. The finalproduct was confirmed as one single band by SDSPAGE and the purity was identified as98.0%and the total recovery yield was up to33.3%.(4) The result of the activity-test in vitro suggested that TFT-GGH, A-GGH and G-GGHcould enhance the production of cAMP. Meanwhile, A-GGH and G-GGH significantlyincreased cAMP levels by10-fold than GGH without N-terminal extension. Meanwhile,A-GGH could efficiently enhance the glucose-lowering effect which was still detectedapparently after the administration for24hours.(5) Molecular mass analysis of A-GGH showed major peaks at6.85104and7.15104m/z. Additionally, a new peak (7.15104m/z), which is close to the theoretical molecularweight (72kD), was observed. The result confirmed that the stability of fusion protein GGHanalogue with N-terminal extension was somewhat improved.