Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry

Abstract

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZRA vector under the control of AOX1 promoter, which included a polyhistidinetag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native Nand C-termini. Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a spectral peak at m/z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu-Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile-Glu-Gly-Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.

Expression of human growth hormone gene in Pleurotus eryngii

“…Basidiomycete mushrooms have not been considered as a platform for the production of therapeutic proteins until now [20]. Previously, several other expression system such as Bacillus subtilis [21], Escherichia coli [22], Pichia pastoris [23], transgenic mice [24], and transgenic goat [25] have been used for producing hGH. This is the first report to express hGH in P. eryngii by the Agrobacterium-mediated transformation system, and we have demonstrated that it was feasible to express a pharmaceutically important gene in mushroom.…”

Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris

“…We hypothesized that the proteolysis was associated with the cargo protein FKBP12 because the Factor Xa sequence has not been proteolyzed by P. pastoris in other published reports [36, 37]. Therefore, two new constructs were designed, which were pJV3 (MBP-FXa-Spe9-myc-His6) and pJV4 (MBP-FXa-EGFP-myc-His6), as illustrated in Figure 1.…”

“…Normally, the Factor Xa site is used after purification to separate the cargo protein from MBP. This protease recognition site (Ile-Glu-Gly-Arg) should not be recognized in P. pastoris [36, 37]. Because of our initial findings, we hypothesized that the Factor Xa site was being recognized by an uncharacterized protease inside the P. pastoris cell and, as a result, the Factor Xa site was replaced with an enterokinase cleavage site.…”

Recombinant snake venom metalloproteinase inhibitor BJ46A inhibits invasion and metastasis of B16F10 and MHCC97H cells through reductions of matrix metalloproteinases 2 and 9 activities

“…Production and purification of recombinant BJ46a rBJ46a protein was produced according to the manufacturer's instructions [10,14]. Briefly, recombinant GS115-BJ46a was amplified to an OD 600 of 4.0 in log-phase growth in 25 ml of BMGY medium [1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base, 0.1 mol/l potassium phosphate buffer pH 6.0, 4 Â 10 -5 % (w/v) biotin, 1% (v/v) glycerol, 4 Â 10 -3 % (w/v) histidine] at 301C using a shaking incubator (300 rpm).…”