Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields.

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts.

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.

Current and prospective applications of metal ion-protein binding.

Since immobilized metal ion affinity chromatography (IMAC) was first introduced, several variants of this method and many other metal affinity-based techniques have been devised. IMAC quickly established itself as a highly reliable purification procedure, showing rapid expansion in the number of preparative and analytical applications while not remaining confined to protein separation. It was soon applied to protein refolding (matrix-assisted refolding), evaluation of protein folding status, protein surface topography studies and biosensor development. In this review, applications in protein processing are described of IMAC as well as other metal affinity-based technologies.

Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones.

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 degrees C in acetic acid. The alpha-and beta-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of > 98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80-90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.

Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells.

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.