Design and production of a novel chimeric human growth hormone superagonist fused to human Fc domain

Abstract

Background and purpose: Growth hormone (GH) has been known as a crucial metabolic hormone expressed at the pituitary and the other number of cells and tissues and responsible for body growth. Because of the short half-life of GH, daily subcutaneous injections were shown to be more effective for GH therapy. This represents a burden for patients. So, there is a strong effort from the industry to create a long-acting form of GH and lots of technologies like GH fusion proteins are used to increase GH half-life.

Experimental approach: In this study, the Fc domain of human IgG1 with serine-glycine linkers was attached to the C-terminal of a GH superagonist via molecular cloning. The presence of recombinant vector in E. coli host was confirmed by PCR. SDS-PAGE and western blot analysis showed the expression of recombinant proteins in the bacterial lysate. The binding ability to growth hormone receptors is determined by ELISA.

Findings / results: Our results showed that the novel SupGH-Fc has a good binding affinity to its receptor in ELISA in comparison to standard GH, although it has a big size.

Conclusion and implications: Our data in this study clearly demonstrated the expression of the SupGH-Fc in a recombinant protein expression system. It is an introduction to the production of the new recombinant GH, which can bind to its receptor more effectively than commercial growth hormones and also might have a longer half-life.

Keywords: Fc fusion proteins; Growth hormone; Growth hormone superagonist; Long half-life.

Fig. 1

Schematic diagram of the expression vector and recombinant proteins. (A) Gene sequence of SupGH with NdeI and BamH1 restriction site; (B) gene sequence of Fc with BamH1 and HindIII restriction site; (C) final gene construct of SupGH-Fc and final structure of the recombinant protein. SupGH, Growth hormone superagonist.

Fig. 2

Schematic diagram of plasmid (pCold-I) and recombinant protein (SupGH-Fc). SupGH, Growth hormone superagonist.

Fig. 3

PCR analysis of original vectors containing SupGH and FC before ligation. Lane 1: DNA ladder, lane 2: PCR product of vector containing Fc at 681 bp, and lane 3: PCR product of vector containing SupGH at 600 bp. SupGH, Growth hormone superagonist; PCR, polymerase chain reaction.

Fig. 4

Colony PCR analysis of Escherichia coli DH5α transformed with pCold-I vector containing SupGH-Fc. Lane 1: DNA ladder, lanes 2, 4, and 5 are those colonies which amplification did not occur. Arrow in lane 3 showed an amplified PCR product of SupGH-Fc with 1281 bp length. Although, in lane 6 amplified PCR product of SupGH-Fc was exist. SupGH, Growth hormone superagonist; PCR, polymerase chain reaction.

Fig. 5

Dot blot analysis of transformed Escherichia coli bl21 expressed SupGH-Fc. A: serial dilution of commercial human GH as a positive control, B: serial dilution of bacterial lysate, and C: serial dilution of culture media. All the digits 1-6 were related to serial dilution respectively: 1/10, 1/100, 1/1000, 1/10000, 1/100000, 1/1000000. SupGH, Growth hormone superagonist.

Fig. 6

SDS-PAGE analysis of pellets and supernatant fractions of transformed Escherichia coli bl-21. Lane 1: protein molecular weight marker (14-116 kDa), lane 2: supernatants after sonication (soluble fraction), lane3: bacterial pellet after sonication (insoluble fraction), lanes 4 and 5: induced with IPTG with dilution 1/50 and 1/10 respectively. Lane 6: uninduced total cell lysate. Arrow indicates the band representing SupGH-Fc. SupGH, Growth hormone superagonist; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Fig. 7

(A) SDS-PAGE that shows the 99% purification of SupGH-Fc, (B) western blot analysis of SupGH-Fc. In both A and B, purified SupGH-Fc was seen around 50 kDa. SupGH, Growth hormone superagonist; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Fig. 8

Sandwich ELISA with SupGH-Fc. ELISA was conducted by plate coated with hGH receptor. Commercial human GH was used as control. The results showed that SupGH-Fc significantly had a high affinity to its receptor compared to human GH as a control group with the same molarity (400 nmol). **P < 0.001 Indicates significant differences compared with the control group. BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; SupGH, growth hormone superagonist.