Relationship Between The Affinity And Biopotency Of Recombinant Human Growth Hormone And Its Receptor

Objective:In order to explore the relationship between the affinity and biopotency of human growth hormone (hGH) interacting with its receptor and to find a high potency hGH analogue, we tried to decrease the affinity of hGH in site 1 and increase the affinity in site 2 with its receptor, promote the homologous dimerization of hGH receptor (hGHR), and improve the hGH biopotency at the same time.Methods:1. Subcloning, expression and purification of the recombinant protein of hGHwt, hGHmut, hGHBP and hGHBPmut.1.1 The gene of the hGHwt was double digested with NdeⅠand HindⅢand then cloned into the expression vector pET20b(+). The mutants including hGH-I4V, R16L, L45D, N109Y, D116F, R167K, S184F, L45D/R64D, R167K/S184F and I4V/E56D/R64M were designed at the hGH site 1 or site 2, and mutated by site-directed mutagenesis. The mutations were confirmed by sequence reaction. The successful recombinations were transformed into the cell strain E.coli. BL21 (DE3), and then the single clone was picked out. The expression of hGHwt and the hGHmut was induced by 1mM Isopropylβ-D-1-thiogalactopyranoside (IPTG). The inclusion body was purified by denaturation, dialysis and ion-exchange column chromatography. The fractions of hGHwt and their mutants were identified by SDS-PAGE and then detected the value of A260 and A280 by the UV spectrometer.1.2 Expression and purification of the recombinant hGHBP, hGHBP-S201C and hGHBP-S237C.The plasmids of pET20b(+)/hGHBPwt, S201C and S237C were transformed into E. coli. BL21(DE3). The expression of hGHBPwt, hGHBP-S201Cand hGHBP-S237C was induced by 1mM IPTG, and then the inclusion body was denatured and refolded. Finally one-step purification is done by Q-sepharose F.F. The purity of hGHBP is analyzed by SDS-PAGE and UV spectrometer.2. Detect the affinity and kinetics of hGHwt and hGHmut binding with hGHBP First, the competitive binding potency of unlabled hGHwt or hGHmut with 125I-hGH to hGHBP was detected by radio-immunology assay (RIA). Second, the binding kinetics of hGHwt or hGHmut with hGHBP was analyzed by biomolecule interaction analysis system (BIAcore).3. Detect the biopotency of hGHmut in vitro and in vivo3.1 Establish the cell line that stably expressing hGHRThe plasmid pcDNA3.1(+) and the hGHR gene were double digested with BamHⅠand XbaⅠrestriction enzymes, and connected with T4 ligase. The plasmid confirmed sequence was transfected into the cell line of BaF3 which proliferation was IL-3 dependent. Via the resistance selection of G418, we picked out one clone that can express hGHR stably. The expression of hGHR in the cell clone was verified by RT-PCR and Western blot.3.2 Test the hGHwt and hGHmut biopotency in vitroThe biopotency of hGHwt and hGHmut was determined by the cell proliferation assay with the hGH dependent BaF3 cell line.3.3 Test the hGHwt and hGHmut bioactivity in vivoThe biological activity of hGHmut was detected by administration of hGHwt and hGHmut to Lewis Dwarf Rat. After four weeks injected of hGHwt and hGHmut, we observed the weight, length, the IG-1 level in serum and the main organs (whole brain, heart, kidney, and liver) of the Lewis Dwarf Rat.4. Analysis the relationship between hGH affinity and its bioactivityCorrelation analysis is used to study the relationship between hGH affinity and its bioactivity promoting the Lewis Dwarf Rat body weight gain. The correlation coefficient, r2, quantifies the direction and magnitude of correlation.Results:1.1 After the sequencing analysis, ten mutants were successful constructed including 14V, R16L, L45D, N109Y, D116F, R167K, S184F, L45D/R64D, R167K/S184F, I4V/E56D/R64M. The results from SDS-PAGE analysis showed only one band can be observed at about 22kDa after purification. The ratio of A(260/280) for hGHwt and hGHmut was 0.5～0.7. The concentration range of hGHwt and hGHmut was from 0.8 to 2mg/ml. 1.2 The results from SDS-PAGE electrophoresis and calculating the ratio of A (260/280) showed the recombinant hGHBP, hGHBP-S201C and hGHBP-S237C with over 95% purity.2. The affinity of hGHwt and hGHmut with their hGHBP2.1 The results from RIA showed that the IC50 of hGHwt, hGHmut-R167K and R167K/S184F binding with hGHBP are 2.18×10-8M·L-1, 3.26×10-9M·L-1, and 6.44×10-9M·L-1 seperately. The affinity of R167K and R167K/S184F with hGHBP was increased up to 6.69-fold and 3.39-fold, compared with hGHwt (***P<0.001). The affinity of hGHmut I4V, L45D, S184F, L45D/R64D and I4V/E56D/R64M is lower than that of hGHwt (*p<0.05). However, there was no significant difference of affinity compared hGHmut R16L, N109Y and D116F with hGHwt.2.2 After the BIAcore assay, we found that the Site1 affinity of hGHmut R167K was improved significantly, but the Sitel affinity of 14V, R16L, L45D, N109Y, D116F, S184F, L45D/R64D, R167K/S184F and I4V/E56D/R64M was decreased with some degree.3. hGHwt and hGHmuts biopotency in vitro and in vivo3.1 The cell line expressing hGHR stably has been establishedThe results from RT-PCR and the Western blot indicated that the cell clone BaF3-hGHR-A15 could express hGHR at a high level and stably.3.2 The biopetency of hGHwt and hGHmuts in vitroWhether hGHmut-R167K with higher affinity on site1 or S184F with lower affinity on site 1 both did not significantly improve the proliferation of BaF3-hGHR-A15 cell comparing with the hGHwt group,*p<0.05; Interesting, hGHmut-N109Y and D116F, could promote BaF3-hGHR-A15 cell proliferation obviously.3.3 Bioactivity of hGHwt and hGHmut in vivoCompared with the hGHwt group, neither the high affinity on site1 of hGHmut R167K, nor the low affinity on sitel of hGHmut L45D, S184F and L45D/R64D had any effect on the body weight of the Lewis Dwarf Rat, p>0.05. However, the hGHmut-N109Y and D116F, similar to the affinity of hGHwt, had a higher biopotency for the Lewis Dwarf Rat.4. The correlation coefficient between affinity on site1 and bioactivity suggested there was no direct relevancy, r2=0.1177.Conclusion:The result from RIA and BIAcore indicated that the binding potency of hGHmut-R167K with hGHBP is higher than that of hGHwt, but its biopotency and bioactivity were not improved whether in vitro or in vivo. Those hGHmuts with the lower affinity on sitel did the same bioactivity to R167K. Interestingly, the biopotency and bioactivity were improved obviously for hGHmut-D116F and N109Y, which displayed the affinity on site1 similar to hGHwt. Our results indicated that:1, it has little correlation between sitel affinity and biological effects for hGHmut and hGHBP; 2, the biopotency and bioactivity of D116F and N109Y, which may be the good candidates of high potency hGH analogue, have been improved remarkably.