Optimization of codon usage
The sequence of optimized hGH gene was deposited in GenBank with the accession numbers of MT321110. Rare gene codons that could reduce translation efficiency were adapted to the expression system of E. coli codon usage. The Codon Adaption Index (CAI) was improved from 0.85 to 0.87. Meanwhile, GC content was also optimized to increase the length of the mRNA half-life and its stability, and the stem-loop structures that blocked the ribosomal connection were removed (Fig. 1 A and Fig. 1 B). FOP of 68 was attained after optimization (Fig.1 C and Fig. 1 D). The ideal percentage range of GC content is between 30-70%. GC content adjustment resulted in the average of 50.59% after optimization (Fig. 1 E and Fig. 1 F).
Design of secretory signal peptide
The designed secretory signal peptide includes three domains. Domain A consists of a region with positively charged amino acids. Domain B consists of a hydrophobic region with a stretch of hydrophobic amino acids and domain C consists of the conserved site for signal peptidase cleavage. The sequence of the natural signal and the designed signal based on the general structure of the natural sec-dependent signal sequences are shown in fig. 2. The altered amino acids have a lower line, and the alignment of these two signals shows their amino acid different.
Our initial studies showed that the natural signal peptide of the protein A is unable to secrete hGH. Therefore, the natural secretory signal was modified and optimized. Our results show that comparing to the natural signal peptide, the modified one became functional in secreting the hGH. The modified signal peptide is able to secrete the recombinant hGH to the both periplasm space and culture medium. As can be seen in Fig. 2, a series of amino acid changes have taken place in different regions of the peptide signal, including the N, H, and C2 regions, which increased the ability of the altered peptide signal to secrete growth hormone out of the cell.
For example, replacing arginine with leucine in the modified form of signal peptide has given it a more positive charge in the N domain and increased its ability to enter the cell membrane which has a negative charge. Another important change is seen in the H domain, in which the amino acid isoleucine replaces threonine and the amino acid serine is changed to alanine in the hydrophobic domain, which, as shown in Fig. 5, increases the hydrophobicity of the H region.
Designed gene and tertiary structure
The schematic view of the pET26 plasmid as well as the designed gene structure is shown in supplementary data file 1. The signal peptide hGH-mutant (jei36c) has two Arginine residues R10 and R12 while the signal peptide of the native hGH has one Argentine residue R10. (Fig. 3).
Signal peptide functional analysis
The results of likelihood score-based predictions for the native and modified jei36c signal peptides are shown in Table 3. For prediction with signal peptide (SP) and tail anchor (TA) datasets, scores were calculated from the probability values of TA and SP models (Table 3 and fig. 4).
Hydrophobicity characteristics of the designed signal peptide
ProtScale online software was used to evaluate the hydrophobicity of the designed signal peptide comparing to the native signal peptide [13] (Fig. 5). Drawing the hydrophobicity and hydrophilicity profiles for these two signal peptides and examining their different domains to observe the changes in their hydrophobicity and hydrophilicity is shown in Fig. 5 (panels E and B). Comparison of these changes shows that the corresponding profiles in these two signal peptides have changed significantly.
The changes in the mutant signal peptide have been able to increase its hydrophilicity of the N region and, conversely, the hydrophobicity of the H region, and ultimately facilitate the secretion of hGH out of the cell. Taken together, these changes increasing the efficiency of this signal peptide in secreting hGH and make it optimized for the secretion of growth hormone from the sec pathway.
Stability of mRNA
To evaluate the stability of the transcribed mRNAs, the mRNA sequences of hGH resulted from fusion of both native and designed signal peptides were submitted to the mfold online server (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form) [14]. During codon optimization, an attempt was made to remove the secondary structure that attenuates or stops mRNA translation, and as it is shown in the fig. 6, the translation inhibitory structure is not seen in the starting region.
Evaluation of recombinant growth hormone expression
Different pET26-based plasmids containing the fusion of various signal peptides to the hGH coding region were transformed into E. coli BL21 (DE3) strain.
Recombinant growth hormone expression was induced under different temperatures as well as various concentrations of glycine and lactose. The following are the optimum conditions which led to the high level of hGH expression in our experiment.
Test conditions A: induction of expression with 10% lactose, 1.5% glycine and induction in OD 1.5, incubation at 30 ° C overnight. The growth hormone bands are shown in the fig. 7.
Test condition B includes induction of expression with 7% lactose, glycine 1% after reaching the bacterial OD to 1.5 and incubation at 25 ° C. in this experiment the sampling time was also reduced to eight hours. In this situation, the supernatant from the bacterial culture was centrifuged at 11000×g for 5 min and the supernatant was precipitated by TCA. As shown in the fig. 8, the hGH bands are present in all supernatants except No. 3.
The expression of recombinant hGH was confirmed by western blotting. As shown in fig. 9A and 9B, Western blot analysis using a specific polyclonal Antibody raised against hGH was used to confirm the expression of the hormone. Arrows in all figures 6-8 represent the expressed recombinant hGH that is processed by the cell secretory system and secreted into the environment.
Protein purification
The expressed recombinant hGH was then purified by affinity chromatography and purification was evaluated using SDS-PAGE analysis. As shown in fig. 10, the hGH is shown as a single, pure band (Line 6, panel B), comparing to its pre-purification state (Lines 1-5, panel A).
Optimization results of the periplasmic hGH expression and secretion
In order to investigate the possible interactions between the factors affecting the production of periplasmic and cytoplasmic hGh production along with their optimal levels, the central composite design (CCD) was used. The matrix is designed and the responses are shown in table 2. Analysis of variance (ANOVA) based on the response surface model for the periplasmic hGH (Table 4) was calculated. Regarding the expression of periplasmic hGH, , the Model F value of 3.23 implies that the model is significant. Values of “Prob > F” less than 0.05, indicate the significance of the model terms. In this case, C: Cell density at induction time (OD.600), and D: Post induction time (h) are significant. Values > 0.1 demonstrate that the model terms are not significant. The “Lack of Fit F value” of 1.42 implies that relative to the pure error, Lack of Fit is not significant. We prefer the model to fit and it is in suitable agreement (Table 4).
Multiple regression analysis of the data was performed, and a first-order polynomial equation for the periplasmic hGH (ug/mL) (Y) according to the coded factors was expressed as below:
Y = 5.89-2.44 A+1.81 B+2.79 C+4.58 D+3.23 AB+1.78 AC+0.027 AD-1.25 BC-1.19 BD-1.68 CD
A, B, C, and D, are coded values for IPTG (mM), temperature (◦C), cell density at induction time (OD600) and post induction time (h), respectively. The above equation can be rewritten as:
Y = -9.72-48.08 A+0.22 B+16.03 C+2.12 D+1.24 AB+8.91 AC+0.01 AD-0.38 BC-0.03 BD-0.56 DC
According to the fig. 11, the optimum conditions for maximum expression of recombinant periplasmic hGH (panels, A and B) in E. col are suggested as follows: IPTG = 0.6mM, Temperature = 26 0C, post induction time = 10h and OD at induction time = 1
Optimization results of the cytoplasmic hGH expression
The designed matrix and the responses are shown in Table 2. Analysis of variance (ANOVA) based on response surface model for cytoplasmic hGH (Table 5) was calculated. For cytoplasmic hGH expression, the Model F value of 4.92 implies that the model is significant. Values of “Prob > F” less than 0.05, show that the model terms are significant. In the case of cytoplasmic expression, C: Cell density at induction time (OD.600), D: Post induction time (h) and interaction between B: Temperature (˚C), and C: Cell density at induction time (OD.600) (BC) are significant model terms. The “Lack of Fit F value” of 0.72 implies that Lack of Fit is not significant relative to the pure error. The “R Squared” of 0.83 is in reasonable agreement (Table 5).
Multiple regression analysis of the data was performed and a first-order polynomial equation for the cytoplasmic hGH (ug/mL) (Y) regarding coded factors was expressed as below:
Y = 27.06 - 4.26A+15B+9.3C+23D + 15AB+3AC+17.5AD-12.75BC+2.99BD-11.75CD
where A, B, C, and D, are coded values for IPTG (mM), temperature (◦C), cell density at induction time (OD600), and post induction time (h), respectively. In the actual variables, the above equation can be rewritten as:
Y = -62.95-251.44A+2B+152.79C+1.34D+5.77AB+15C+7.29AD-3.92BC+0.077BD-3.92d
According to the fig. 11, the optimum conditions for maximum cytoplasmic recombinant hGH expression (panels, C and D) in E. col were suggested as IPTG = 0.6mM, temperature = 26 0C, post induction time 10h and OD at induction time = 1
Fig. 12 shows the interactions between temperature and OD at induction time (OD600) at IPTG = 0.6mM and post induction time 10h for cytoplasmic hGH expression.
Determination of functional hGH concentration
After generating the standard curves for each test by plotting the absorbance versus the concentration of each controls, concentrations of the active form of the hGH were obtained from the standard curve (all row data were reported in supplementary data file 2). The results showed that the amount of active form of hGH produced in the periplasm was about 28.12% of the total proteins, while this amount was equal to 10% for the cytoplasm fraction. These results indicate that the secretion of hGH into the periplasmic space by this method causes the protein to fold into its proper structural conformation.