Characterisation of a secreted form of recombinant derived human growth hormone, expressed in Escherichia coli cells.
Recombinant DNA derived human growth hormone (rhGH), Genotropin, has been expressed in E. coli cells as a pre-hormone, where the heat stable enterotoxin II signal peptide (STII) was linked to hGH to get secretion of the hormone to the periplasmatic space. The pre-hormone was efficiently cleaved during secretion, by an endogenous signal peptidase generating the correct N-terminal (Phe) end as shown by protein sequence analysis. The purity of rhGH was studied by SDS-PAGE, in combination with laser densitometry and HI-HPLC. These techniques showed that the level of modified rhGH forms, e.g. aggregated and proteolytically cleaved (16 and 6 kDa) in the preparation was in the 0.5-1% range. Furthermore, evidence that the correct disulphide bonds (Cys53-Cys165; Cys182-Cys189) were formed in rhGH during secretion has been shown by a combination of tryptic fingerprint and amino acid analysis. CD-spectroscopic analysis suggested an identical secondary structure to that of pituitary derived human growth hormone (pit-hGH). Isoelectric focusing revealed an isoelectric point (pI) for rhGH of 5.0 similar to pit-hGH and in excellent agreement with the theoretical value 5.1, based on the primary sequence. Finally, an apparent molecular weight of 22,000 was obtained for rhGH, by SDS-PAGE. All these physico-chemical studies suggest that rhGH is structurally identical to pit-hGH, somatotropin.
Gellerfors
P
P
Kabi Vitrum Peptide Hormones AB, Stockholm, Sweden.
Eketorp
G
G
Fhölenhag
K
K
Pavlu
B
B
Johansson
S
S
Fryklund
L
L
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D013006:Growth Hormone / Q000032:analysis* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000494:pharmacology
D000596:Amino Acids; D004220:Disulfides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D013006:Growth Hormone